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R Eritja

Publications and source records attributed to R Eritja.

At least 55 records · Page 3Linked to original sources
PubMed1996

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2-nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.

Carbamates↗
PubMed1995

Divalent zinc cations induce the formation of two distinct homoduplexes of a d(GA)20 DNA sequence.

Homopurine DNA sequences are highly structurally polymorphic. In particular, d(GA)n DNA sequences are known to be capable of forming intramolecular foldbacks, bimolecular homoduplexes, and tetrastranded complexes. Counterions play a determinant role on the equilibria between the different structural conformers of d(GA)n sequences. In this paper, the effect of divalent zinc cations on the structure of a d(GA)20 oligonucleotide has been analyzed by CD spectroscopy and polyacrylamide gel electrophoresis. Depending on the precise experimental conditions at which zinc is added, two distinct conformations of the d(GA)20 oligonucleotide are stabilized. At neutral pH in the absence of zinc, d(GA)20 is partially organized into intramolecular foldbacks and bimolecular homoduplexes [Casasnovas et al. (1993) J. Mol. Biol. 233, 671-681]. Under these conditions, addition of zinc results in the stabilization of the bimolecular homoduplex which is nonspecific for zinc since it is also stabilized by divalent magnesium cations, increasing ionic strength, or decreasing pH. Its CD spectrum is identical to that reported earlier for parallel-stranded d(GA)n homoduplexes [Rippe et al. (1992) EMBO J. 11, 3777-3786]. On the other hand, if zinc is added under conditions where the d(GA)20 oligonucleotide is exclusively single-stranded, a different bimolecular homoduplex appears which is only observed in the presence of zinc. The zinc-specific duplex melts cooperatively, and, in contrast to the nonspecific duplex, its thermostability is high. Transition from the nonspecific to the zinc-specific duplex is observed at high zinc concentrations or at high temperatures. The transition is cooperative. These results are discussed in the context of the specific cation effects on the formation of intramolecular R.R.Y triplexes at d(GA.TC)n DNA sequences.

Animals↗
PubMed1995

Dam methylase from Escherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates.

We have measured steady-state kinetics of the N6-adenine methyltransferase Dam Mtase using as substrates non-selfcomplementary tetradecamer duplexs (d[GCCGGATCTAGACG]-d[CGTCTAGATCC-GGC]) containing the hemimethylated GATC target sequence in one or the other strand and modifications in the GATC target sequence of the complementary strands. Modifications included substitution of guanine by hypoxanthine (I), thymine by uracil (U) or 5-ethyl-uracil (E) and adenine by 2,6-diamino-purine (D). Thermodynamic parameters were obtained from the concentration dependence of the melting temperature (Tm) of the duplexes. Large differences in DNA methylation of duplexes containing single dI for dG substitution of the Dam recognition site were observed compared with the canonical substrate, if the substitution involved the top strand (on the G.C rich side). Substitution in either strand by uracil (dU) or 5-ethyluracil (dE) resulted in small perturbation of the methylation patterns. When 2,6-diamino-purine (dD) replaced the adenine to be methylated, small, but significant methylation was observed. The kinetic parameters of the methylation reaction were compared with the thermodynamic free energies and significant correlation was observed.

2-Aminopurine↗
PubMed1995

Commensalism, adaptation and gene flow: mosquitoes of the Culex pipiens complex in different habitats.

Two ecotypes have been described for Culex pipiens mosquitoes of the temperate zone: a human commensal type and a feral type, but their degree of evolutionary differentiation and taxonomic status are still unclear. The commensal form is characterized by life-history traits probably adaptive to underground man-made environments. This situation has sometimes been considered as an example of recent speciation although the existence of intermediate forms indicates that the balance between gene flow and disruptive selection should first be assessed. The present study was concerned with (1) the determination of biological traits involved in adaptation to commensalism, and (2) the pattern of gene flow within and between ecotypes in a restricted area. It was found that (1) significant differences in biological traits exist between mosquitoes from different habitats, (2) characteristics of the commensal type are not universal in mosquitoes from underground man-made habitats, (3) allozyme markers do not clearly differentiate ecotypes and (4) insecticide resistance genes, which reveal recent migration, occur in each ecotype. These results are discussed in the context of possible speciation due to commensalism.

Adaptation, Physiological↗
PubMed1995

Digoxigenin-labeled phosphorothioate oligonucleotides: a new tool for the study of cellular uptake.

The mechanisms and intracellular pathways by which many oligonucleotide analogs enter cells to exert the desired antisense effects are not fully understood and remain a matter of debate. In this study, we describe the synthesis of 5'-digoxigenin-labeled phosphorothioate oligonucleotides and show their use to examine intracellular oligonucleotide distribution within Epstein-Barr virus-transformed B cells. Comparison of digoxigenin-labeled and fluorescein-labeled oligonucleotide distribution shows the same intracellular fate, suggesting that digoxigenin modification does not interfere with intracellular routing. Double immunofluorescence studied by conventional fluorescence and confocal microscopy with antibodies to the labeling molecule and to lysosome-associated membrane protein indicate that oligonucleotides mainly accumulate in the lysosomal compartment. Digoxigenin labeling offers an alternative to study oligonucleotide uptake and distribution by immunoelectron microscopy. Two different approaches have been studied: immunogold labeling in heavily fixed and resin-embedded cells and immunogold labeling in lightly fixed and cryoultramicrotomy processed cells. The results confirm the major lysosomal accumulation of digoxigenin-labeled oligonucleotides and demonstrate that the antigenic capacity of digoxigenin is not damaged by any of the procedures used. Therefore, the conjugation of the functionalized digoxigenin molecule at the 5' end of phosphorothioate oligonucleotides provides a new tool in the study of oligonucleotide uptake and intracellular distribution at both cellular and ultrastructural levels.

B-Lymphocytes↗
PubMed1995

Kinetic studies of MvaI DNA methyltransferase interaction with modified oligonucleotide duplexes.

We have measured steady-state kinetics of a N4-cytosine methylase, M.MvaI, using as substrates modified non-selfcomplementary tetradecanucleotide duplexes containing the CCWGG target sequence. The inner or outer localisation of the dI residue in the MvaI recognition site seems to be of little importance since the specificity constants kcat/KM are only 2 to 7 fold smaller than that of the canonical substrate. Replacement of dG residues by dI in both strands resulted in a 25 to 60-fold decrease of the specificity constant. Modifications of the phosphate backbone or opening of the sugar ring of one of the dG residues had only little influence on the action of M.MvaI. The enzyme appears to be rather tolerant to different kinds of modifications in its substrate in the mainor groove.

Base Sequence↗
PubMed1995

Efficacy of ground ultra-low volume larvicidal treatments using temephos and Bacillus thuringiensis var. israelensis against Culex pipiens larvae.

Spray drift determinations of truck-mounted ULV applications of temephos and Bacillus thuringiensis var. israelensis (B.t.i.) in simulated field trials were conducted in the Baix Llobregat area near Barcelona, Spain, using larval Culex pipiens as bioindicators. A 50:50 temephos:oil mixture applied at 350 ml/min, while traveling 9 kph, resulted in 100% control of larvae 10-50 m from the point of application (with the exception of 82% mortality at 30 m); control decreased rapidly to 50% at 55 m. Mortality due to B.t.i. application ranged from 92 to 100% within 12 m, and generally decreased therafter to about 11% at 30 m.

Animals↗
PubMed1994

Functional regulation of platelet/endothelial cell adhesion molecule-1 by TGF-beta 1 in promonocytic U-937 cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a widely distributed cell adhesion molecule present on monocytes, macrophages, and monocytic cell lines. Treatment of the promonocytic cell line U-937 with TGF-beta 1 induces homotypic cellular aggregations, simultaneous with an increase in surface expression and specific transcripts of PECAM-1. The TGF-beta-induced cell adhesion phenomena are not dependent on LFA-1, intercellular adhesion molecule-1 (ICAM-1), very late Ag-4 (VLA-4), or very late Ag-5 (VLA-5). However, the phenomena seem to be directly mediated by PECAM-1 because 1) it is inhibited by the addition of Abs or an antisense oligonucleotide specific for PECAM-1; and 2) TGF-beta 1-treated U-937 cells bind to PECAM-1-expressing mouse transfectant fibroblasts, but not to mock transfectants. In addition, this aggregation phenomena are divalent cation-dependent and requires the integrity of the cytoskeleton. Analysis of the intracellular signaling pathways indicates that TGF-beta 1 induces protein kinase C activity, as well as PECAM-1 phosphorylation and association with cytoskeletal components. Furthermore, in this model, an autocrine mechanism for releasing the bioactive form of TGF-beta 1 operates, allowing PECAM-1 activation. These results provide evidence that TGF-beta 1 regulates PECAM-1 function by increasing the expression and activating the adhesion of PECAM-1 in monocytic cells. These two mechanisms seem to be necessary for adhesion because independent inhibition of either expression or activation of PECAM-1 leads to abrogation of cellular aggregation.

Antigens, Differentiation, Myelomonocytic↗
PubMed1994

Nucleotide insertion and primer extension at abasic template sites in different sequence contexts.

Efficiencies of insertion and extension at a single site-directed abasic lesion, X, were measured while varying 5'- and 3'-template bases adjacent to X. The preference for insertion was found to be A > G > T approximately C, with the "upstream" (3'-neighboring) template base perturbing insertion efficiencies by an order of magnitude or more. Efficiencies of synthesis past the abasic lesion depended strongly on the "downstream" (5'-neighboring) template base and on the properties of the polymerase. HIV-1 RT favored "direct" extension of X.A > X.G > X.T > X.C, by addition of the next correct nucleotide. However, it was found that X.C, least favored for direct extension, was most favored for "misalignment" extension, occurring when the DNA structure in the vicinity of the lesion collapsed to realign a primer 3'-C terminus opposite a downstream template G site. Polymerase properties have an important role in copying abasic lesions. Drosophila DNA polymerase alpha, HIV-1, and AMV reverse transcriptases had "little" difficulty inserting opposite abasic lesions, with efficiencies comparable to misinsertions opposite normal template bases. However, AMV RT did not extent past the lesion using direct or misalignment mechanisms. Wild-type and mutant T4 DNA polymerases were used to show that although exonucleolytic proofreading inhibits lesion bypass, the presence of a highly active proofreading exonuclease is not sufficient to prevent bypass.

DNA Damage↗
PubMed1994

Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase.

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.

2-Aminopurine↗
PubMed1994

Three-dimensional crystal structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor-groove-binding drug Hoechst 33258.

The molecular structure of the DNA A-tract dodecamer d(CGCAAATTTGCG) complexed with the drug Hoechst 33258 has been determined by X-ray diffraction analysis. The Hoechst molecule binds in the DNA minor groove covering the sequence AATTT of the central A-tract, with the piperazine group close to one of the GC regions. The drug molecule makes two three-centered hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and O2 atoms of the DNA bases. Although a high propeller twist is observed in the A-tract, only one unsymmetrical three-centered hydrogen bond is present in the DNA major groove. The structure is compared with other minor-groove-binding drug complexes and the influence of these drugs on DNA A-tracts is discussed.

Base Sequence↗
PubMed1994

Chemical synthesis of a fully active transcriptional repressor protein.

Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.

Amino Acid Sequence↗
PubMed1993

Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV-1 reverse transcriptase.

Deoxyribonucleotide insertion efficiencies were measured opposite site-directed abasic template lesions using human immunodeficiency virus 1 reverse transcriptase (HIV-1RT), and the efficiencies to continue primer synthesis beyond the lesion, by addition of the "next correct" deoxynucleotide, were measured as a function of sequence context. Insertion of purines was favored over pyrimidines, A > G > T approximately C. Primer extension past the lesion occurred by two distinct mechanisms, either by direct or by misalignment extension. An "A-rule" appeared to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In misalignment extension, the primer terminus is realigned from a site directly opposite the lesion to a new position opposite a neighboring template base 5' to the lesion. Direct extension efficiencies were measured in 16 different configurations, by varying 4 bases at the primer 3'-termini and 4 at the 5'-side (downstream) of the lesion. The predominant order of direct extension was A > G > T approximately C, similar to that observed for insertion. Reduced primer extension rates were not caused by a reduction in HIV-1 RT-DNA binding. Primers terminating in C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment extension efficiencies were 27:1, 2.5:1, and 1:25 for A, G, and C opposite the lesion, respectively. For the case of primers terminating in T, misalignment extension was not observed. A striking result was that while primers were extended past an abasic lesion by HIV-1 RT in both direct and misalignment modes, avian myeloblastosis virus RT failed to catalyze significant extension by either mode.

Animals↗
PubMed1993

Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine.

To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.

Base Sequence↗
PubMed1993

Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence.

The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.

Adenosine Triphosphate↗
PubMed1992

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.

Autoradiography↗