Base Pair Sites Research Articles

Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3–22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed ≥2.3% sequence divergence between taxa and ≤1.3% within taxa. We developed a PCR-RFLP based method for unequivocally distinguishing amongst the four cryptic European taxa of this subgenus, B. cryptarum, B. lucorum, B. magnus and B. terrestris, and used it to analyse 391 females of the former three species collected across Ireland, all of which could be unambiguously assigned to species. Bombus lucorum was the most widely distributed and abundant of the cryptarum–lucorum–magnus species complex, comprising 56% of individuals, though it was significantly less abundant at higher altitudes (>200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

Alanine Scanning and Fe-BABE Probing of the Bacteriophage ø29 Prohead RNA–Connector Interaction

The DNA packaging motor of the Bacillus subtilis bacteriophage ø29 prohead is comprised in part of an oligomeric ring of 174 base RNA molecules (pRNA) positioned near the N termini of subunits of the dodecameric head–tail connector. Deletion and alanine substitution mutants in the connector protein (gp10) N terminus were assembled into proheads in Escherichia coli and the particles tested for pRNA binding and DNA–gp3 packaging in vitro. The basic amino acid residues RKR at positions 3–5 of the gp10 N terminus were central to pRNA binding during assembly of an active DNA packaging motor. Conjugation of iron(S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) to residue S170C in the narrow end of the connector, near the N terminus, permitted hydroxyl radical probing of bound [32P]pRNA and identified two discrete sites proximal to this residue: the C-helix at the junction of the A, C and D helices, and the E helix and the CE loop/D loop of the intermolecular base pairing site.

Site-Specific DNA Photocleavage by Rhodium Intercalators Analyzed by MALDI-TOF Mass Spectrometry

The DNA photocleavage reaction of mismatch-selective Rh complexes has been analyzed by MALDI-TOF mass spectrometry as well as gel electrophoresis analysis of radioactively tagged oligonucleotides. Analogous results are obtained with these two techniques showing site-specific cleavage neighboring the mismatch to yield primarily 5'- and 3'-phosphate termini. Additional intermediates and products are observed, however, using MALDI-TOF analysis. MALDI-TOF mass spectrometry is seen to be particularly powerful in the analysis of DNA cleavage by site-specific molecules. The method requires no radioactive labeling, only little material, and analysis can be accomplished within minutes. Moreover, this mass spectral analysis of DNA cleavage yields direct information regarding products rather than simply the base pair site of cleavage.

Kinetics of Photoinduced RNA Refolding by Real‐Time NMR Spectroscopy

By introducing a photolabile group in the Watson-Crick base-pairing site of a guanosine residue, a bistable 20-base RNA sequence was forced into a less stable conformation. A single laser pulse released the native sequence, and the subsequent refolding equilibration was monitored with time-resolved NMR spectroscopy. This permitted a quant. description of the refolding process. [on SciFinder (R)]

Selective production of orthoalkyl phenols on Cu 0.5Co 0.5Fe 2O 4: a study of catalysis and characterization

Phenol alkylation was carried out on Cu 1− x Co x Fe 2O 4 ( x = 0–1) by employing various alkylating agents: CH 3OH, CH 3OCOOCH 3 dimethyl carbonate (DMC), C 2H 5OH, 2-C 3H 7OH and isobutene (IB), under a wide variety of reaction conditions; a comparative analysis is made. Cu 0.5Co 0.5Fe 2O 4 displays the highest phenol alkylation activity, irrespective of the alkylating agents. The catalytic conversion of phenol follows the following order: MeOH ≈ DMC > EtOH > isopropanol ≈ IB. Ortho-dialkylated product formation decreases as the chain length of alkylating agent increases. X-ray photoelectron spectra (XPS) and Auger electron spectroscopy analysis of fresh and spent Cu 0.5Co 0.5Fe 2O 4 catalysts revealed a partial reduction of metal ions due to reaction. Valence band studies reveal an increase in the overlap of metal ion 3d bands from fresh to spent catalysts due to a large decrease in the energy gap between them. The high activity observed is attributed to an optimum distribution of Cu-species with hetero-atom neighbors on the surface and a large overlap between the Cu and Co 3d bands. A mechanism involving the reaction of phenol and alcohol molecules adsorbed simultaneously on the same acid–base pair site is supported by IR spectroscopy results from the reactants and pyridine adsorbed on catalyst.

Cooperative binding of tetrameric p53 to DNA.

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.

FTIR study of unsupported molybdenum sulfide—in situ synthesis and surface properties characterization

Self-supporting molybdenum sulfide samples with high surface area were prepared by in situ thermal decomposition in vacuum of ammonium thiomolybdate. BET specific surface area of the resulting material has its highest values of about 120 m 2/g for samples treated in situ at 473 K and decreases on further heating, while the composition of samples treated at 423–473 K is close to MoS 3, and after heating up to 573 K corresponds to MoS 2. FTIR spectra show that precursor decomposition is almost completed already after treatment at 423 K, although the sample still contains admixed ammonium ions. After 473 K most of contaminations are gone. Spectra of adsorbed NH 3, ND 3, CO, SO 2, CO 2, COS and CH 3SH adsorbed on samples pretreated at 423–473 K are very close to those obtained for air-contacted MoS 2 powder. However higher band intensities and the use of labeled compound enabled us to identify the bands of coordinately bonded NH 3 molecules and to point to the existence of acidic sites capable to protonate ammonia. Molecularly adsorbed CH 3SH could be distinguished from its dissociative form; this reveals the existence of acid–base pair sites on bulk molybdenum sulfide.

Oxidative Dehydrogenation of Ethylbenzene over Cu1-xCoxFe2O4Catalyst System: Influence of Acid–Base Property

A series of Cu–Co ferrites with the general formula Cu1-xCoxFe2O4 (x = 0, 0.25, 0.50, 0.75 and 1.0) was prepared by a low-temperature hydroxide coprecipitation route. The catalyst systems were characterized by adopting various physicochemical techniques. The acid–base properties were studied in detail, and the catalytic activity as well as the selectivity for oxidative dehydrogenation of ethylbenzene was compared for various compositions. FTIR adsorption of pyridine is carried out to understand the relative acidity of various compositions of the systems. IR studies of spinel surface with adsorbed CO2 and adsorption studies of electron acceptors such as 7,7,8,8-tetracyanoquinodimethane, 2,3,5,6-tetrachloro-1-4-benzoquinone and p-dinitrobenzene are carried out to evaluate the nature of basic sites and the strength and distribution of electron donor sites present on the spinel surface. It is found that acidity (basicity) of the Cu1-xCoxFe2O4 spinel system increases (decreases) from x = 0 to 1. A good correlation was found between the activity for this reaction and the surface acid–base properties of the catalysts. Intermediate compositions show better catalytic performance, among which x = 0.50 is superior and demonstrates an intermediate acid–base character. It was observed that dehydrogenation of ethylbenzene to styrene proceeds mainly on an acid–base pair site.

Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3'-terminal psoralen on a third strand directed to the mutant base pair.

Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell beta-globin gene mutation and the comparable wild-type beta-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3'-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established.

Oxide-catalyzed conversion of acetic acid into acetone: an FTIR spectroscopic investigation

Adsorptive and catalytic interactions of gas phase acetic acid with surfaces of alumina, titania and ceria were observed by in situ Fourier-transform infrared (FTIR) spectroscopy on heating from room temperature up to 400 °C. The results revealed that, on alumina the acid was irreversibly, non-dissociatively adsorbed in the form of hydrogen-bonded molecules, and dissociatively in the form of bidentate bound acetate species over the full range of temperature scanned. In the gas phase, no chemical change was observed. On titania, the gas phase remained unchanged on heating up to 300 °C, but at 400 °C the acetic acid was largely converted into acetone (as well as CO 2 and H 2O) and minor products of isobutene and methane. Similar changes to the gas phase were observed on ceria, however, at 300 °C. The acetic acid conversion on ceria was almost complete. The appearance of acetone molecules in the gas phase was pertained by the emergence of IR absorptions implying the formation on the surface of unsaturated carbonyl species. Thus, the formation of these surface species, together with isobutene and CH 4 molecules in the gas phase, was considered consequent to the occurrence of further surface reactions of acetone molecules. Observance of structural and chemical stability of the surface and bulk of TiO 2 and CeO 2 throughout the reaction was taken to emphasize the catalytic nature of the acetic acid conversion into acetone in the gas phase, a process that is evidently more simple and economic than the conventional pyrolytic synthesis of acetone from metal acetate compounds in the solid state. The catalytic sites were suggested to be Lewis acid–base pair sites, with the Lewis acid sites (Ti 4+ or Ce 4+) being reducible. Mechanistic pathways were proposed for the observed adsorptive and catalytic interactions of acetic acid molecules on the test oxides.

Genotyping of DNA Using Sequence-Specific Methyltransferases Followed by Immunochemical Detection

Modern molecular genetics relies on the ability to map the positions of genes on chromosomes, relative to known DNA markers. The first such DNA markers described were Restriction Fragment Length Polymorphisms, but any restriction endonuclease used for RFLP mapping is just one member of a restriction-modification pair. For each restriction endonuclease, there is a companion methyltransferase (MTase) that has the same DNA sequence specificity. Therefore, in principle, it should be possible to use MTases rather than restriction enzymes to detect polymorphic sites in DNA. We have used sequence-specific DNA MTases to detect genetic polymorphisms in closely related viral pathogens. If at least one MTase recognition site is present in PCR-amplified DNA, then methyl groups are incorporated; if no MTase site is present, then methyl groups are not incorporated. When several different sequence-specific DNA MTase reactions are carried out, the pattern of methyl incorporation defines a DNA MTase genotype. DNA MTase Genotyping (DMG) can be used to rapidly diagnose heritable or infectious diseases, to immunochemically detect DNA at defined 2 to 8 base pair sites, or to characterize the amplicons by constructing ordered maps.

DNA/lipid complex organogel with shape‐memory behavior

AbstractDNA and amphiphilic N,N,N‐trimethyl‐N‐hexadecylammonium bromide (THAB) were stoichiometrically mixed together to produce a DNA–lipid complex, which was dissolved in dimethyl sulfoxide (DMSO) at 65°C and then crosslinked with isophorone diisocyanate (IPDI). The obtained organogel swelled reversibly in DMSO, responding to the variation of temperature. Interestingly, we observed that the gel exhibited a temperature‐dependent shape‐memory behavior. Above 65°C, whatever the shape the gel was deformed to, it could retain the new shape as the temperature was decreased to room temperature, while, when the gel was heated to 65°C again, it could recover its initial shape. The shape‐memory characteristic is supposedly originated from the transition between the close‐packed and the destroyed DNA/lipid conformation. The elastic urethane crosslinking bonds between base‐pair sites act as a fixing phase. The gel holds promise in its application as a gentle actuator. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 259–263, 2002

Vapor-Phase Alkylation of Toluene with Isopropanol over Mg/Al, Ni/Al and Cu/Al Hydrotalcites (Layered Double Hydroxide)

Vapor-phase isopropylation of toluene has been carried out over Mg/Al, Ni/Al and Cu/Al calcined hydrotalcites (CHTs). Reaction conditions were optimized for alkylation by varying temperature, weight hourly space velocity and reactant mole ratio. Side-chain alkylation is found to be more predominant than ring alkylation over Mg/Al CHT, while Ni/Al and Cu/Al CHTs favored ring alkylation. The main products were isobutylbenzene and cymene. The combined participation of acidic and basic sites of the materials is found to be crucial for both side-chain and ring alkylation of toluene with isopropanol. An X-ray diffraction pattern of the resulting oxide indicates a diffuse MgO structure. The incorporation of small amounts of Al3+ to MgO generates new surface Lewis acid--base pair sites.

The activation of CD4 for H/D exchange over H-zeolites

The H/D exchange was studied between CD4 and H-zeolites using pulsed a microcatalytic reactor and MS analysis. Methane-d3 was the only exchange product, while a large fraction of the reactant was retained by the zeolite. The apparent activation energies (Ea) were determined for CHD3 production assuming first-order reaction kinetics. The Ea was close to 80 kJ mol-1 over the H-ZSM-5 and H-mordenite, and about 50 kJ mol-1 over H-beta. Results suggested that, at the low methane loading applied, the H/D exchange was initiated predominantly by heterolytic dissociation of CD4 to CD3δ- and Dδ+ over Lewis acid-Lewis base pair sites.

Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion.

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson-Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N(2),N(2)-dimethylguanosine, N(6),N(6)-dimethyladenosine (m(6)(2)A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N(2)-methylguanosine (m(2)G) or N(4)-methylcytidine. A borderline case is represented by N(6)-methyladenosine (m(6)A). Although generally a duplex-preserving modification, our data indicate that m(6)A in specific strand positions and at low strand concentrations is able to effectuate duplex-hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm(2)GGm(6)(2)Am(6)(2)AGGUC. In analogy, it is demonstrated that the tandem m(6)(2)A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson-Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

Selective O-methylation of catechol using dimethyl carbonate over calcined MgAl hydrotalcites

Methylation of catechol with dimethyl carbonate as an alkylating agent has been carried out over calcined MgAl hydrotalcites in the temperature range 523–623 K. Guaiacol and veratrole were obtained as the major products, along with small amounts of catechol carbonate and C-alkylated products. At 573 K, the total O-selectivity was 96.1% with a guaiacol selectivity of 84%. At higher DMC/catechol molar ratios and longer contact times, guaiacol selectivity decreased markedly with a concomitant increase in the selectivity of veratrole. An optimum value of the strength of acid–base pair sites involved in the adsorption of DMC and catechol makes hydrotalcite-based oxides better catalysts compared to alumna and basic MgO. It is possible that the nature of alkylating agent also can affect the product selectivity along with the experimental factors and the acid–base properties of the catalysts.

Catalytic transfer reduction (CTR) of alkyl alkyl, alkyl aryl, cyclic and unsaturated ketones over calcined Mg–Al hydrotalcites

Calcined Mg–Al hydrotalcites are very efficient catalysts in the vapor phase transfer reduction of a wide variety of ketones to afford corresponding alcohols. Aryl alkyl ketones were converted to aryl alkenes in excellent yields at 548 K. The catalytic activity of calcined hydrotalcites is attributed to the co-existence of acid–base pair sites, with strong basic sites, which can promote transfer hydrogenation.

Photocleavage of DNA by 4'-bromoacetophenone analogs.

4'-Bromoacetophenone analogs, which are able to generate monophenyl radicals capable of hydrogen atom abstraction, were investigated as possible photoinducible DNA cleaving agents. The potential of 4'-bromoacetophenone as a possible new DNA cleaver is explored. Pyrrolecarboxatmid conjugated 4'-bromoacetophenones, in particular, DNA cleaving activity and sequence-selectivity on the contiguous AT base pair sites.

Ortho-alkylation of phenol derivatives with methanol over magnesium oxide catalysts. 1. Characterization of promoted magnesium oxide catalysts

Cr- and Pt-promoted magnesium oxide catalysts were used to catalyze ortho-alkylation of phenol derivatives in the presence of excess methanol. The characteristic properties of the MgO surface measured by XRD, SEM, BET, and TPD suggest that Cr exists as fine chromium oxide particles on the MgO surface, while a Pt and MgO interaction causes an increase of the BET surface area as well as of surface acid and base amounts. Cr/MgO shows a specialty for ortho-alkylation of phenol derivatives with methanol without forming O-alkylated products, i.e., anisole or methylated anisole. The catalytic activity and the stability of Cr/MgO are improved by the addition of a fairly small amount of Pt. The effect of Pt can be explained by the Pt and MgO interaction which increases both the acid and base amounts. A mechanism involving the reaction of phenol derivatives and methanol adsorbed simultaneously on acid–base pair sites is proposed.