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PubMedDate not supplied

Theoretical studies of cis-Pt(II)-diammine binding to duplex DNA.

The binding of cis-Pt(II) diammine (cis-DP) to double-stranded DNA was studied with several kinked conformations that can accommodate the formation of a square planar complex. Molecular mechanics (MM) calculations were performed to optimize the molecular fit. These results were combined with quantum mechanical (QM) calculations to ascertain the relative energetics of ligand binding through water vs direct binding of the phosphate to the ammine and platinum, and to guide the selection of DNA conformations to model complex formation. Based on QM and MM calculations, models are proposed that may be characterized by several general features. A structure involving hydrogen bonding between each ammine and distinct adjacent phosphate groups, referred to as closed conformation (CC), has already been reported. This is also found in the crystal structure of small dimers. We report alternative conformations that may be important in platination of duplex DNA. They are characterized by an intermediate conformation (IC), involving hydrogen bonding between one ammine and phosphate group, and an open conformation (OC), without ammine phosphate hydrogen bonding. The IC and OC can be stabilized by water bridges in the space between the ammine and the phosphate groups. Sugar puckers alternate from the type C(2')-endo or C(1')-exo (S), to the type C(3')-endo or C(2')-exo (N), with intermediate types near O(1')-endo (O). In general, the sugar puckers alternate from S to N to S through the platinated region (3'-TpG*pG*p-5'), with the complexed strand exhibiting, (3')-S*-N*-S-(5') alternation, while the complementary strand shows either (3')-S*-N*-S-(5') or (3')-S*-N*-O-(5') alternation. In both the OC and IC, a hydrogen bond is found between the ammine and O4(T) on thymine (T) at the (3') end, adjacent to the complex site. There is a continuous range of backbone conformations through the platinated region which relate the OC to the IC. The models presented suggest that the dynamics of the binding of the cis-Pt(II)-diammines to adjacent N7(G) in double-stranded DNA may encompass several conformational possibilities, and that water bridges may play a roll in supporting open and intermediate conformations. Proton-proton distances are reported to assist in the experimental determination of conformations.

Cisplatin↗

PubMed2006

Nonsense-mediated mRNA decay mutation in Aspergillus nidulans.

An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5' nonsense codons are more effective at triggering NMD than 3' nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3' exon-exon junction.

Adaptor Proteins, Signal Transducing↗

PubMed1986

Proposals for the mu-active conformation of the enkephalin analog Tyr-cyclol(-N gamma-D-A2-bu-Gly-Phe-Leu-).

The conformational behavior of the sterically restricted cyclic peptide Tyr-cyclo(-N gamma-D-A2-bu-Gly-Phe-Leu-), proposed recently as an enkephalin analog with high opiate activity, is examined by theoretical investigations. The method used allows the search of conformational energy minima associated with cyclic structures fitting a hypothetical opiate pharmacophore. The results obtained show that, despite the fact that many cyclic structures of low conformational energy can be found for this compound, only one of them can be retained as a conformer presenting the characteristic features of the imposed pharmacophore. This conformation is stabilized by an intramolecular H-bond between the D-A2bu-carbonyl and the Leu NH group so that a beta-turn is formed. This structure also presents a high mobility of the Tyr1 side-chain which can fit the tyramine moiety of rigid opiates with minor loss of conformational energy. A two-step binding mechanism is proposed for the interactions of this cyclic peptide with its receptor which could be an intermediate between the "zipper" model proposed for flexible linear peptides and the "lock-and-key" model adapted to rigid molecules. The selectivity of enkephalin analogs for mu and delta opioid receptors is discussed in light of the present theoretical investigations.

Enkephalin, Leucine↗

PubMed1992

Stability of septohippocampal neurons following excitotoxic lesions of the rat hippocampus.

The present study examined the effects of removing hippocampal nerve growth factor (NGF)-producing neurons upon cholinergic and noncholinergic septohippocampal projecting neurons. To deplete septal/diagonal band neurons of their intrinsic source of NGF, rats received unilateral intrahippocampal injections of ibotenic acid and were sacrificed 2-24 weeks later. Choline acetyltransferase and parvalbumin immunohistochemistry failed to reveal changes in the number of cholinergic or gamma-aminobutyric acid-containing neurons, respectively, within the septal/diagonal band region ipsilateral to the hippocampal lesion at any time point examined. Additionally, immunocytochemical localization of nonphosphorylated and phosphorylated neurofilament proteins did not reveal abnormal staining characteristics within the septal/diagonal band complex, suggesting that this lesion does not alter cytoskeletal features of neurons which project to the hippocampus. Selected rats received unilateral hippocampal lesions and 3 months later were injected with fluorogold into the remaining hippocampal remnant and with wheat germ agglutinin conjugated to horse radish peroxidase into the intact contralateral hippocampus. Both retrograde tracers were predominantly transported to their respective ipsilateral septum and vertical limb of the diagonal band. This indicates that following the lesion, septal/diagonal band neurons still project ipsilaterally and sprouting to the NGF-rich contralateral side does not occur. RNA blot analysis revealed a decrease in NGF mRNA expression within the lesioned hippocampus with a maximum reduction of approximately 70%. In contrast, no change in NGF mRNA expression was observed within the ipsilateral septum relative to the contralateral side. The present study demonstrates that removal of hippocampal target neurons does not alter the number, morphology, or projections of both cholinergic and noncholinergic septal/diagonal band neurons.

Animals↗

PubMed1986

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species.

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.

Archaea↗

PubMed2005

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase.

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Brönsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.

Chlorpromazine↗

PubMed2005

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study.

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.

2-Naphthylamine↗

PubMed2004

A comparative study of adduct formation between the anticancer ruthenium(III) compound HInd trans-[RuCl4(Ind)2] and serum proteins.

Formation of adducts between the antitumor ruthenium(III) complex [HInd]trans-[RuCl(4)(Ind)(2)] (KP1019) and the plasma proteins serum albumin and serum transferrin was investigated by UV-vis spectroscopy, for metal-to-protein ratios ranging from 1:1 to 5:1. In both cases, formation of tight metal-protein conjugates was observed. Similar spectroscopic features were observed for both albumin and transferrin derivatives implying a similar binding mode of the ruthenium species to these proteins. Surface histidines are the probable anchoring sites for the bound ruthenium(III) ions in line with previous crystallographic results. In order to assess the stability of the KP1019-protein adducts the influence of pH, reducing agents and chelators was analysed by UV-vis spectroscopy. Notably, there was no effect of addition of EDTA on the UV-vis spectra of the conjugates. The pH-stability was high in the pH range 5-8. Experiments with sodium ascorbate showed that there was just some alteration of selected bands. The implications of the present results are discussed in relation to the pharmacological behavior of this novel class of antitumor compounds.

Animals↗

PubMed2003

SCA7 knockin mice model human SCA7 and reveal gradual accumulation of mutant ataxin-7 in neurons and abnormalities in short-term plasticity.

We targeted 266 CAG repeats (a number that causes infantile-onset disease) into the mouse Sca7 locus to generate an authentic model of spinocerebellar ataxia type 7 (SCA7). These mice reproduced features of infantile SCA7 (ataxia, visual impairments, and premature death) and showed impaired short-term synaptic potentiation; downregulation of photoreceptor-specific genes, despite apparently normal CRX activity, led to shortening of photoreceptor outer segments. Wild-type ataxin-7 was barely detectable, as was mutant ataxin-7 in young animals; with increasing age, however, ataxin-7 staining became more pronounced. Neurons that appeared most vulnerable had relatively high levels of mutant ataxin-7; it is interesting, however, that marked dysfunction occurred in these neurons weeks prior to the appearance of nuclear inclusions. These data demonstrate that glutamine expansion stabilizes mutant ataxin-7, provide an explanation for selective neuronal vulnerability, and show that mutant ataxin-7 impairs posttetanic potentiation (PTP).

Animals↗

PubMed2002

From natural products to clinically useful antifungals.

In our search for natural products with a broad spectrum of antifungal activity as lead compounds for novel treatments for mycoses, we have isolated echinocandin-type lipopeptide FR901379 and lipopeptidolactone FR901469, as novel water-soluble antifungal agents that inhibit the synthesis of 1,3-beta-glucan, a key component of the fungal cell wall. Since the cell wall is a feature unique to fungi and is not present in nonfungal eukaryotic cells, inhibitors of the synthesis of fungal cell wall components such as 1,3-beta-glucan have potential for selective toxicity to fungi and not to the host. In this short review, we describe efforts directed at synthetic modification of FR901469 and FR901379 with the ultimate goal of identifying new entities with suitable profiles as development candidate compounds. The main thrust of our work to date has been replacement of the highly flexible lipophilic side chains of the natural products with a view to reducing the hemolytic potential associated with these compounds, and to enhance chemical stability and/or in vivo antifungal efficacy. As a result of these efforts, we recently discovered a novel analog, FK463 (micafungin). Micafungin is currently in phase III clinical trials worldwide as a parenteral agent for various mycoses, and a new drug application (NDA) was recently filed in Japan.

Anti-Bacterial Agents↗

PubMed2004

Surface-tunable photoluminescence from block copolymer-stabilized cadmium sulfide quantum dots.

The static and time-resolved photoluminescence properties of polystyrene-b-poly(acrylic acid) (PS-b-PAA)-stabilized cadmium sulfide quantum dots (CdS QDs) have been characterized for the first time, demonstrating tunable emission spectra and quantum yields via different chemical treatments of the PAA layer. Samples with the PAA layer in its cadmium carboxylate form showed more-intense band-edge emission and relatively high quantum yields compared with samples in which the PAA layer was in its acid form. This activation effect is explained in terms of passivation of trap sites on the QD surface by specific interactions between the QD and the cadmium-neutralized PAA layer. Lifetimes of band-edge and trap state emission for the various samples ranged from 40 to 61 ns and 244 to 360 ns, respectively. Impressive long-term stability was also shown for a sample of cadmium-neutralized PS-b-PAA-stabilized QDs dispersed in toluene, which maintained 90% of its photoluminescence over 57 days aging under ambient conditions. It is also shown that Cd2+ activation of photoluminescence does not occur when Mg2+ ions are added to similar QD solutions, indicating potential of these block copolymer-stabilized QDs as Cd2+-selective sensors. Irrespective of chemical treatment of the PAA layer, the external PS brush layer effectively stabilized all samples in various organic solvents, resulting in clear CdS colloids with no observed precipitation over several months. Dynamic light scattering and gel permeation chromatography revealed differences in the aggregation numbers and hydrodynamic radii of colloidal QDs for different treatments of the PAA layer, attributed to the lower solubility of the poly(cadmium acrylate) blocks compared to the PAA blocks in the acid form. Finally, it was demonstrated that the PS-b-PAA-stabilized QDs could be well dispersed in PS homopolymer, producing optically transparent photoluminescent films which retained the emission features of the colloidal QDs. Stable and surface-tunable optical properties via the PAA layer and polymer solubility and processability via the PS layer make these PS-b-PAA-stabilized CdS QDs exciting "building blocks" for the bottom-up assembly of functional hierarchical materials for photonics, sensors, and bio-labeling applications.

Journal Article↗

PubMed2006

Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP.

The nicotinic acetylcholine receptor (nAChR) is the prototype member of the superfamily of pentameric ligand-gated ion channels. How the extracellular ligand-binding domain coordinates selective binding of ligand molecules to different subtypes of the receptor is unknown at the structural level. Here, we present the 2.2-A crystal structure of a homolog of the ligand-binding domain of the nAChR, Aplysia californica AChBP (Ac-AChBP), in complex with alpha-conotoxin ImI. This conotoxin is unique in its selectivity toward the neuronal alpha3beta2 and alpha7 nAChR, a feature that is reflected in its selective binding to Ac-AChBP compared with other AChBP homologs. We observe a network of interactions between the residues of the ligand-binding site and the toxin, in which ImI Arg-7 and Trp-10 play a key role. The toxin also forms interactions in the ligand-binding site that were not seen in the complex of Ac-AChBP with PnIA(A10L D14K), a conotoxin variant that lacks binding selectivity to AChBP homologs. In combination with electrophysiological recordings obtained by using the wild-type alpha7 nAChR and L247T mutant, we show that conotoxin ImI inhibits ion conduction by stabilizing the receptor in a desensitized conformation. Comparison of the Ac-AChBP-ImI crystal structure with existing AChBP structures offers structural insight into the extent of flexibility of the interface loops and how their movement may couple ligand binding to channel gating in the context of a nAChR.

Amino Acid Sequence↗

PubMed2004

Oxytalan fibres in the periodontal ligament of equine molar cheek teeth.

The distribution and arrangement of oxytalan fibres were examined in periodontal specimens of cheek teeth from seven horses. Oxidation prior to aldehyde fuchsin exposition permitted a selective staining of the oxytalan fibres, which are a distinct component of the elastic fibre system. On three horizontal levels of the periodontium--(a) subgingival, (b) middle third and (c) apical--two oxytalan fibre groups were shown histologically: 'blood vessel-related' and 'independent' oxytalan fibres. In levels a and b, both groups were arranged in a typical occluso-apical alignment along the reserve crown. Single oxytalan fibres deviated from their general course in order to attach to the cementum. In these cemental entheses the oxytalan fibres ran parallel to collagen fibre bundles. The interpretation of such morphological features emphasized the oxytalan fibres' capacity to improve the stability of periodontal blood vessel walls during masticatory movements. Level c, especially in regions next to the persisting epithelial root sheath, is the site of oxytalan fibre generation. This is a prerequisite for the facilitation of periodontal regeneration and reorganization during dental growth and eruption.

Animals↗

PubMed2005

Spectroscopic and kinetic properties of the horseradish peroxidase mutant T171S. Evidence for selective effects on the reduced state of the enzyme.

Studies on horseradish peroxidase C and other haem peroxidases have been carried out on selected mutants in the distal haem cavity providing insight into the functional importance of the distal residues. Recent work has demonstrated that proximal structural features can also exert an important influence in determining the electronic structure of the haem pocket. To extend our understanding of the significance of proximal characteristics in regulating haem properties the proximal Thr171Ser mutant has been constructed. Thr171 is an important linking residue between the structural proximal Ca2+ ion and the proximal haem ligand, in particular the methyl group of Thr171 interdigitates with other proximal residues in the core of the enzyme. Although the mutation induces no significant changes to the functional properties of the enzyme, electronic absorption and resonance Raman spectroscopy reveal that it has a highly selective affect on the reduced state of the enzyme, effectively stabilizing it, whilst the electronic properties of the Fe(III) state unchanged and essentially identical to those of the native protein. This results in a significant change in the Fe2+/Fe3+ redox potential of the mutant. It is concluded that the unusual properties of the Thr171Ser mutant reflect the loss of a structural restraint in the proximal haem pocket that allows 'slippage' of the proximal haem ligand, but only in the reduced state. This is a remarkably subtle and specific effect that appears to increase the flexibility of the reduced state of the mutant compared to that of the wild-type protein.

Horseradish Peroxidase↗

PubMed1990

General features in the stoichiometry and stability of ionophore A23187-cation complexes in homogeneous solution.

Existing literature describing the stoichiometry and stability of complexes between A23187 and divalent cations in solution has been extended to include additional transition series cations, the heavy-metal cations Cd2+ and Pb2+, plus seven lanthanide series trivalent cations. Stability constants of 1:1 complexes between the ionophore and the divalent cations vary by 6.2 orders of magnitude between Cu2+ and Ba2+ which are the strongest and weakest complexes, respectively. Considering alkaline-earth and first-series transition cations together, the pattern of stability constants obeys the extended Irving-Williams series as is seen with many nonionophorous liganding agents. Cd2+ and Pb2+ are bound with an affinity similar to those of Mn2+ and Zn2+, whereas the lanthanides are bound with little selectivity and slightly higher stability. Titration of the ionophore in the 10(-5) M concentration range with di- and trivalent cations gives rise first to complexes of stoichiometry MA2 and subsequently to MA as the metal concentration is increased. The second stepwise stability constants for formation of the MA2 species exceeds the first constant by approximately 10-fold. With lanthanides, heavy metals, and transition-metal cations, OH-, at near physiological concentrations, competes significantly with free ionophore for binding to the 1:1 complexes. This competition is not apparent when Ca2+ or Mg2+ are the central cations. Possible implications of the 1:1 complex selectivity pattern, the ionophore-hydroxide competitive binding equilibria, and potential ternary complexes involving 1:1 ionophore:cation complexes and other anions present in biological systems are discussed with respect to the ionophore's transport selectivity and biological actions.

Biological Transport↗

PubMed2006

From bench to bedside--preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A).

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A) is just the second ruthenium-based anticancer agent after NAMI-A which was developed to the stage of clinical trials. Important steps in the mode of action of KP1019 are thought to be the binding to the serum protein transferrin and the transport into the cell via the transferrin pathway. Additionally, the selective activation by reduction in the tumor might contribute to the low side effects observed in in vivo studies. Apoptosis is induced at non-toxic levels via the mitochondrial pathway. These features distinguish it from the established platinum anticancer drugs and suggest that different types of cancer might be treatable with this drug. Indeed, promising activity against certain types of tumors, which are not successfully treatable with cisplatin, and only a very low incidence of acquired resistance has been observed in in vitro and in vivo studies. Recently, a clinical phase I trial was finished in which none of the treated patients experienced serious side effects, while disease stabilization in five of six evaluable patients was achieved. In this review, the preclinical and early clinical development of KP1019 - from bench to bedside - is recapitulated.

Antineoplastic Agents↗

PubMed2003

Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1.

Proteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1 beta and tumor necrosis factor alpha. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1.

Amino Acid Sequence↗

PubMed1996

Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus.

We have studied a novel class of DNA sequences that cause DNA polymerases to pause. These sequences have the central consensus Py-G-C and are not necessarily adjacent to hairpins in the DNA template. Since most consensus sequences do not cause pauses under standard conditions, additional template features must exist that make it difficult to incorporate nucleotides at these positions. We believe that these pauses result from constraints that make the conformation change involved in nucleotide selection more difficult. These pauses can obscure parts of DNA sequencing ladders and prevent DNA amplification by the polymerase chain reaction. The addition of betaine, and some related compounds, relieves these pauses.

Bacteriophage T7↗