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M Buckle

Publications and source records attributed to M Buckle.

35 records · Page 2Linked to original sources
PubMed1996

The polypyrimidine tract binding (PTB) protein interacts with single-stranded DNA in a sequence-specific manner.

Polypyrimidine tract binding (PTB) protein is a cellular factor whose function is unknown. Various RNA or single-stranded DNA sequences have been shown to interact with PTB. In this paper, using laser UV crosslinking and electrophoretic mobility shift assays to probe DNA-protein interactions, we demonstrate that PTB binding at a single-stranded DNA target is highly sequence-specific. We provide data showing that PTB interacts with the top strand of the adenovirus major late promoter transcriptional initiator, a sequence rich in pyrimidine residues. We also demonstrate that PTB is organised into at least two different binding domains.

Adenoviridae↗
PubMed1996

Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance.

A rapid direct assay for polymerase-induced elongation along a given template is an obligate requirement for understanding the processivity of polymerization and the mode of action of drugs and inhibitors on this process. Surface plasmon resonance can be used to follow the association and the dissociation rates of a given reverse transcriptase on DNA.RNA and DNA.DNA hybrids immobilized on a biotin-streptavidin surface. The addition of nucleotides complementary to the template strand produces an increase in the local mass, as deduced from an increase in the measured signal, due to elongation of the primer strand that allows an estimation of both the extent and rate of the polymerization process. The terminator drug 3'-deoxy-3'-azidothymidine triphosphate completely abolishes the increase in signal as would be expected from an inhibition of elongation. This technique provides a sensitive assay for the affinities of different polymerases for specific templates and for the effects of terminators of the elongation process.

Bacterial Proteins↗
PubMed1996

The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits a marked preference for Mn(II) rather than Mg(II).

In this investigation, we examine the interaction between the human immunodeficiency virus type I integrase and oligonucleotides that reflect the sequences of the extreme termini of the viral long terminal repeats (LTRs). The results of gel filtration and a detailed binding density analysis indicate that the integrase binds to the LTR as a high-order oligomer at a density equivalent to 10 +/- 0.8 integrase monomers per 21-base pair LTR. The corresponding binding isotherm displays a Hill coefficient of 2, suggesting that the binding mechanism involves the cooperative interaction between two oligomers. This interaction is quite stable, exhibiting a prolonged half-life (t1/2 approximately 13 h) in the presence of Mn2+ cations. Complexes were less stable when formed with Mg2+ (t1/2 approximately 1 h). The role of Mn2+ appears to be in the induction of the protein-protein interactions that stabilize the bound complexes. In terms of the 3'-end processing of the LTR, similar catalytic rates (kcat approximately 0.06 min-1) were obtained for the stable complex in the presence of either cation. Hence, the apparent preference observed for Mn2+ in standard in vitro integration assays can be attributed entirely to the augmentation in the DNA binding affinity of the integrase.

Base Sequence↗
PubMed1994

Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs.

New base-substituted analogs of dCTP containing an azido group have been synthesized and applied to a selective photoaffinity modification of HIV-RT (p66/p51 heterodimer). The labeling of only the 66 kDa subunit of HIV-RT was detected when the enzyme was first irradiated with the analogs and then template (5'-(d)GGTTAAATAAAATAGTAAGAATGTATAGCCCCTACCA-3') and 5' 32P end-labeled 3'-(d)TTACATATCGGGGATGGT-5' primer were added. The 5' 32P end-labeled primer elongated by dCTP analogs in the presence of both HIV-RT and DNA template is able to modify both subunits of HIV-RT and DNA template. This way of specific cross-linking to both DNA (RNA) template and HIV-RT opens up new possibilities to study the HIV-RT active site.

Affinity Labels↗
PubMed1993

E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.

A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.

Carrier Proteins↗
PubMed1993

Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions.

Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.

Base Sequence↗
PubMed1992

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.

Biological Transport↗
PubMed1992

DNA deformation in nucleoprotein complexes between RNA polymerase, cAMP receptor protein and the lac UV5 promoter probed by singlet oxygen.

Singlet oxygen (1O2), generated by exciting an eosin-Tris complex with a high intensity beam of radiation at 532 nm, was used to chemically modify bases in fragments of DNA containing the lac UV5 promoter in the presence of the DNA binding proteins, RNA polymerase and CRP (cAMP receptor protein). Subsequent treatment with piperidine selectively cleaved the DNA at specific modified bases in the sequence. Using this technique we show first that the reactivity of DNA bound by CRP differs in the presence and absence of RNA polymerase. Hence the local conformation of CRP-bound DNA must change during the transition to the open complex. However, no reactivity is observed at the sites of the 40 degrees kinks described in the cocrystal structure (Steitz, 1990). Secondly we show that there is unique CRP-dependent reactivity at a specific site (position -46 on the upper strand) in the open complex. Finally, in the open complex, 1O2 also reacts with sites 90 bp upstream from the transcription start point. This reactivity is qualitatively CRP-independent. We infer that 1O2 reacts at sites where the promoter DNA is significantly distorted, and suggest that the pattern observed reflects the functional orientation of an active transcriptional complex in which the DNA is bent to form an extended loop.

Cyclic AMP Receptor Protein↗
PubMed1991

Protein-DNA cross-linking at the lac promoter.

We report the results of photo-cross-linking of RNA polymerase and the cyclic AMP receptor protein (CRP) to the lac UV5 promoter region carried on either a linear fragment or a supercoiled plasmid. We have devised a protocol that allows the localisation of bases in contact with the protein. RNA polymerase makes contacts within the -10 and -35 regions of the promoter, essentially on the non-template strand. The CRP contact points found in a binary complex are affected by the formation of the ternary complex containing RNA polymerase. Supercoiling has no effect on the position of contacts in any of the complexes. These conclusions were derived from experiments performed using a generally applicable, non-interfering technique that reveals direct contacts between proteins and nucleic acids in nucleoprotein complexes.

Base Sequence↗
PubMed1989

Fine mapping of DNA single-stranded regions using base-specific chemical probes: study of an open complex formed between RNA polymerase and the lac UV5 promoter.

We have used diethyl pyrocarbonate (DEP), which carbethoxylates adenine bases, and dimethyl sulfate (DMS), which methylates guanine residues and single-stranded cytosines, to probe bases in open complexes between RNA polymerase and the lac UV5 promoter in vitro. We compared the kinetics of reactivity between bases in an open complex and those in a single-stranded 35-mer fragment corresponding to the lower template strand of lac UV5 in the region -25 to +10 relative to the transcription start site. We observed that cytosine and adenine residues in the 35-mer fragment reacted according to a second-order process with DMS and DEP, respectively, at sufficiently low concentrations of the reagents and that the degree of reactivity was base position independent. In an open complex in the absence of substrates, we observed reactivity with DEP in adenines from -12 to +4 as well as +21 on the template strand and methylation by dimethyl sulfate of cytosines -6, -4, -2, and -1. No hyperreactivity was observed on the nontemplate strand. The degree of reactivity of bases between -12 and +4 was position dependent, maximum reactivity being displayed by bases in the middle of the region. The reaction was first order within the range of reagent concentration investigated. It was confirmed that in the presence of ApA and UTP cytosine +5, as well as cytosines -6, -4, -2, and -1, in an open complex became reactive to DMS. With regard to DEP the extent of reactivity of the adenine at position +3 was increased markedly, adenine +4 was brought into the single-stranded region, and the overall reactivity of adenine -10 decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1986

Regulation of ATP hydrolase activity of the F0-F1 complex of rat-liver mitochondria during early hepatic regeneration.

Submitochondrial particles prepared from rat liver in the early phase of hepatic regeneration possess a reduced F1 content with respect to F0 in intact F0F1-H+-ATPase complexes. Analysis of ATP hydrolysis showed a significant difference in both ESMP and isolated F1 with regard to the higher affinity Km values (Km,1) obtained from Eadie-Hofstee plots. Both ESMP and F1 from regenerating rat liver showed much lower apparent Km,1 values (0.04 and 0.03 mM, respectively) than the corresponding controls (0.08 mM for both ESMP and F1). Data presented here show that the residual F1 moieties have an altered kinetic pattern with regard to the competitive inhibitor adenosine 5'-[beta,gamma-imido]triphosphate (K1 ESMP from regenerating rat liver = 0.67 microM, K1 ESMP from control rat liver = 2.03 microM). This difference in affinity for [beta,gamma-imido]-ATP is also seen in isolated F1 (K1 regenerating rat liver = 0.04 microM, K1 control rat liver = 0.22 microM). These data indicate that during the disruptive retrodifferential phase of hepatic regeneration, changes at the level of surviving F1 sectors of the F0-F1 ATPase may play a physiological role in preventing ATP hydrolysis in vivo in the brief period of low delta microH+, induced by the presence of non-F1-associated F0 proton-conducting pathways.

Adenylyl Imidodiphosphate↗
PubMed1986

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver.

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the FoF1 ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (delta mu H+) generated by succinate-driven respiration. In control rat-liver submitochondrial particles containing the FoF1 moiety of the ATPase complex, anaerobic relaxation of delta mu H+ showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin and N,N-dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1-Fo ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin-sensitivity-conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat-liver mitochondrial membranes. The results reported show that the structural and functional integrity of the Fo-F1 ATPase of rat liver is severely perturbed during regeneration.

Adenosine Triphosphatases↗
PubMed1985

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver.

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.

Animals↗
PubMed1983

Characterization of a Chinese hamster ovary cell line resistant to uncouplers.

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.

Adenosine Triphosphatases↗
PubMedDate not supplied

Real time in vitro analysis of transcription by RNA polymerase on immobilized DNA fibres.

We have used surface plasmon resonance (SPR) to follow variations in the concentrations of binary complexes as RNA polymerase moves into a transcriptionally competent initiation complex with immobilized DNA fibres containing promoter sequences. The use of SPR to follow complex binding phenomena is described. We have also followed the changes in the mass of initiation complexes following addition of the nucleotide triphosphates prerequisite for transcription on the immobilized template. These signals are interpreted in terms of the escape of RNA polymerase into elongation mode and the subsequent synthesis of nascent RNA molecules.

DNA↗