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D J Clark

Publications and source records attributed to D J Clark.

At least 73 records · Page 4Linked to original sources
PubMed1992

A nucleosome core is transferred out of the path of a transcribing polymerase.

We have determined the fate of a nucleosome core on transcription. A nucleosome core was assembled on a short DNA fragment and ligated into a plasmid containing a promoter and terminators for SP6 RNA polymerase. The nucleosome core was stable in the absence of transcription. The distribution of nucleosome cores after transcription was examined. The histone octamer was displaced from its original site and reformed a nucleosome core at a new site within the same plasmid molecule, with some preference for the untranscribed region behind the promoter. These observations eliminate several models that have been proposed for transcription through a nucleosome core. Our results suggest that a nucleosome core in the path of a transcribing polymerase is displaced by transfer to the closest acceptor DNA.

Animals↗
PubMed1992

Measurement technique for the determination of blood oxygen saturation.

A new optical measurement technique based on spectral scanning is described for the determination of oxygen saturation of whole blood. The principles of this technique are outlined, together with a calibration procedure used to test its feasibility in vitro. The preliminary results show that the accuracy of the new technique is of the order +/- 2% over the full range of oxygen saturation. This degree of accuracy is comparable with commercial CO-oximeters.

Blood Gas Monitoring, Transcutaneous↗
PubMed1992

Endocrine deficit after fractionated total body irradiation.

Endocrine function was assessed in 31 children (17 boys) after fractionated total body irradiation used in the preparative regimen for bone marrow transplantation. Endocrine dysfunction was present in 25 children. Fifteen of 29 had growth hormone insufficiency 0.9-4.9 years after total body irradiation, yet only three of the 15 had received previous cranial irradiation. Five of 30 had thyroid dysfunction: two with a low thyroxine and raised thyroid stimulating hormone (TSH) concentration and three with a raised TSH and normal thyroxine concentration. Thus the incidence of thyroid dysfunction (16%) is much lower than that reported after single fraction total body irradiation (39-59%). In only two children were abnormalities of the hypothalamic-pituitary-adrenal axis demonstrated. The majority of pubertal children assessed (n = 15) showed evidence of gonadal damage. All the pubertal girls (n = 5) had ovarian failure, although there was evidence of recovery of ovarian function in one girl. All seven boys in late puberty showed evidence of damage to the germinal epithelium, and two of three in early puberty had raised follicle stimulating hormone concentrations. Despite the use of a fractionated total body irradiation regimen, endocrine morbidity is substantial and children undergoing such procedures will require long term endocrine review and management.

Adolescent↗
PubMed1991

Histone contributions to the structure of DNA in the nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.

Animals↗
PubMed1991

Superhelical stress and nucleosome-mediated repression of 5S RNA gene transcription in vitro.

Nucleosomes were assembled on a plasmid carrying a Xenopus somatic 5S RNA gene prepared at different superhelix densities. The gene was preferentially assembled into a positioned nucleosome which was stable to superhelical stress. No evidence for a conformational change in the nucleosome was found, even under extreme negative superhelical stress. Transcription in an extract from Xenopus oocyte nuclei was repressed to a degree which depended on the number of nucleosomes assembled. Topoisomerase activity in the extract was effectively inhibited by camptothecin, which had no effect on transcription. Transcription of reconstitutes remained repressed relative to naked plasmids, and was independent of superhelix density. Transcripts from reconstitutes were derived solely from nucleosome-free genes. Thus, a histone octamer positioned on the gene was sufficient to block its transcription. Tryptic removal of the core histone tail domains had no effect on transcription at any superhelix density. Transcription of reconstitutes containing H3/H4 tetramers was also repressed, but not eliminated (unlike reconstitutes containing octamers), and repression was independent of superhelix density. We suggest that removal of histones H2A/H2B from the nucleosome facilitates activation of transcription in the extract. We conclude that superhelical stress alone does not activate transcription of a 5S RNA gene assembled into a nucleosome in vitro.

Animals↗
PubMed1991

Formation of nucleosomes on positively supercoiled DNA.

A transcribing RNA polymerase is thought to generate positive supercoils in front of the advancing transcription complex and negative supercoils behind. We have examined the possibility that positive supercoils might destabilize nucleosomes, facilitating transcription. We show that histone octamers bind to positively supercoiled DNA, and that after the complex is relaxed, 'classical' nucleosomes are present. We tested the possibility that nucleosomes on positively supercoiled DNA are in an altered (presumably more open) conformation, but revert to the classical structure only on release of this stress. However, circular dichroic spectra, and chemical cross-linking and modification of core histones, all suggest that the complexes initially formed on positively supercoiled DNA are classical nucleosomes. Although such structures are stable, their formation requires the plasmid to become more positively supercoiled, resulting in greater superhelical stress. In contrast, formation of nucleosomes on negatively supercoiled DNA relieves superhelical stress. In an exchange experiment in which equilibrium is achieved, nucleosomes transfer from positively to negatively supercoiled DNA, as predicted from the super-coiling free energies of the reactions. This suggests a mechanism for transcription of a gene assembled into chromatin, in which octamers are sequentially transferred from the region in front of the polymerase to the region behind.

Animals↗
PubMed1991

Familial antithrombin III deficiency and Mycoplasma pneumoniae pneumonia.

A 10 year old girl presented with a massive femoral vein thrombosis associated with Mycoplasma pneumoniae pneumonia. Subsequently type I familial antithrombin III deficiency was diagnosed. It is suggested that prophylactic measures aimed at preventing thrombosis should be considered in acutely ill subjects with infection and familial thrombophilia.

Antithrombin III Deficiency↗
PubMed1990

Chromatin assembly on replicating DNA in vitro.

Replicating single-stranded DNA is preferentially assembled into chromatin in Xenopus egg extracts relative to non-replicating double-stranded DNA. We have examined the molecular basis of this phenomenon. Single-stranded DNA itself is not a favored template for nucleosome assembly in comparison to double-stranded DNA. Complementary strand synthesis is required for the rapid assembly of nucleosomes. We present evidence that the assembly of chromatin on replicating DNA is a two step phenomenon. The first step involves the replication of DNA and the assembly of an intermediate structure, the second step involves the sequestration of histones H2A/H2B onto DNA. Histones H2A/H2B are preferentially sequestered onto replicated DNA in comparison to non-replicated DNA incubated in the extract.

Animals↗
PubMed1990

Induction of a calcium/calmodulin-dependent phosphodiesterase during phytohemagglutinin-stimulated lymphocyte mitogenesis.

A calmodulin (CaM)-dependent phosphodiesterase activity that hydrolyzes both cGMP and cAMP was observed in anion exchange high performance liquid chromatography (HPLC) profiles from phytohemagglutinin-stimulated mononuclear cells but not in profiles from unstimulated cells. A single polypeptide was detected by an antibody to the calmodulin-dependent phosphodiesterases on a Western blot of homogenates of stimulated mononuclear cells. The phosphodiesterase activity was immunoadsorbed in a calcium-dependent manner by an antibody to calmodulin but not by an antibody to the 61-kDa bovine brain phosphodiesterase. The mononuclear cell enzyme eluted from the HPLC column in the same fractions as the 63-kDa calmodulin-dependent isozyme from bovine brain and appeared to have the same subunit molecular weight when probed on a Western blot. The electrophoretic mobility of proteolytic fragments derived from the mononuclear cell phosphodiesterase were identical to those from the 63-kDa brain isozyme. The enzyme could be detected in mononuclear cells by activity assays and on a Western blot 14 h after stimulation with mitogen. The enzyme remained elevated for at least 100 h after stimulation. A dose-response experiment with phytohemagglutinin demonstrated that similar concentrations of mitogen could induce both mitogenesis and the phosphodiesterase. The induction of this enzyme requires mRNA as well as protein synthesis but not DNA synthesis. An enzyme similar to the 63-kDa phosphodiesterase found in brain seems to demonstrate a regulatory interface for the metabolism of calcium and cyclic nucleotides during lymphocyte mitogenesis.

Animals↗
PubMed1990

Electrostatic mechanism of chromatin folding.

We describe a theoretical analysis of cation binding in the nucleosome, and in chromatin as it folds, using Manning's polyelectrolyte theory. The theory accounts remarkably well, even quantitatively, both for the interaction of histone charges with DNA in chromatin, and for the essential features of the folding process. The degree of chromatin folding under different ion conditions is reliably predicted by the electrostatic free energy of DNA in the H1 binding site, which determines repulsions between linker DNA segments thus limiting how closely they may approach. The electrostatic free energy is a function of the ionic strength and the residual (unneutralized) DNA charge. Monovalent cations effect chromatin folding primarily by screening the residual charge whilst divalent or trivalent cations bind to DNA reducing its residual charge. The binding of H1 to the linker DNA considerably reduces its electrostatic free energy by displacing bound cations and reducing the residual charge. Thus, native chromatin folds at lower salt concentrations than does H1-depleted chromatin. We conclude that the mechanism of chromatin folding is primarily electrostatic in nature. In vivo ion conditions are such that chromatin is compact but H1 molecules are able to exchange freely, probably due to a low degree of salt-induced dissociation. When H1 molecules exchange, transient local disruptions may occur in the chromatin filament due to repulsion of temporarily H1-free linker DNA from within the filament, such that chromatin "breathes". Thus, the cell can maintain its chromatin in a compact form and access to DNA for sequence-specific DNA-binding proteins and the transcription machinery is still possible.

Animals↗
PubMed1990

Pressure measurements during automatic breast compression in mammography.

Breast compression during X-ray mammography results in improved image quality at a lower radiation dose to the patient, and, as a consequence, the Department of Health recommends that automatic breast compression devices are fitted to mammographic X-ray units. However, the degree of breast compression is not standardized and can vary depending on the size of the patient, the particular mammography X-ray unit and the conditions of its use. A pressure measuring system was used to determine accurately the pressure on the breast. This system takes the form of a fluid-filled neonatal cuff connected to a pressure transducer by a fluid line. The pressure measuring system was calibrated and tested, first without and then with the patients, to assess its practical feasibility. The elements of the pressure measuring system, the techniques involved in its calibration and its use on patients in the clinical environment are described here. The system has proved to be a quick and simple method of relating the pressure on the breast to the pressure reading of the mammography X-ray unit.

Automation↗
PubMed1989

Propranolol induced bradycardia in tetralogy of Fallot.

When an 18 month old girl who had tetralogy of Fallot and episodes of severe cyanosis with loss of consciousness was treated with propranolol there was some improvement. But when the dose was increased she had further episodes of near syncope. Holter monitoring showed extreme intermittent bradycardia with pauses of up to 2.6 seconds. The episodes of near syncope and the bradycardia resolved after propranolol was stopped. Apparent failure of propranolol treatment may on rare occasions be related to drug induced bradycardia rather than to continued episodes of severe cyanosis.

Bradycardia↗
PubMed1988

Differences in the binding of H1 variants to DNA. Cooperativity and linker-length related distribution.

A study of the complexes formed between short linear DNA and three H1 variants, a typical somatic H1, and the extreme variants H5, from chicken erythrocytes, and spH1 from sea urchin sperm, has revealed differences between H1, H5 and spH1 that have implications for chromatin structure and folding. 1. All three histones bind cooperatively to DNA in 35 mM NaCl forming similar, but not identical, rod-like complexes. With sufficiently long DNA the complexes may be circular, circles forming more easily with H5 and spH1 than with H1. 2. The binding of H5 and spH1 to DNA is cooperative even in 5 mM NaCl, resulting in well-defined thin filaments that appear to contain two DNA molecules bridged by histone molecules. In contrast, H1 binds distributively over all the DNA molecules in 5 mM NaCl, but forms short stretches similar in appearance to the thin filaments formed with H5 and spH1. Rods appear to arise from the intertwining of regular thin filaments containing cooperatively bound histone molecules on raising the NaCl concentration to 35 mM. 3. The compositions of the rods correspond to one histone molecule for about every 47 bp (H1), 81 bp (H5) and 112 bp (spH1), suggesting average spacings of 24 bp (H1), 41 bp (H5) and 56 bp (spH1) in the component thin (double) filaments. Strikingly, these values are proportional to the linker lengths of the chromatins in which the particular H1 variant is the main or sole H1.

Animals↗
PubMed1988

Sigma elements are position-specific for many different yeast tRNA genes.

We determined the DNA sequence of seventeen sigma elements and flanking regions in order to investigate the extent of the association between the yeast repetitive element, sigma, and tRNA genes. Fifteen of seventeen sigma elements analyzed begin at position -19 to -16 with respect to the 5' end of a tRNA-coding sequence. This region is close to the initiation point of tRNA gene transcription and contains a sequence which is modestly conserved for a number of tRNA genes. Two pairs of identical sigma elements occur as the long terminal repeats of a sequence which, together with flanking sigma elements, has the structural properties of a retrotransposon; this element has been named Ty3 (manuscript submitted). Hybridization analysis of yeast chromosomal DNA separated by orthogonal field alternation gel electrophoresis (OFAGE) showed that Ty3 and isolated sigma elements are distributed over many chromosomes in the yeast genome.

Base Sequence↗
PubMed1988

A yeast sigma composite element, TY3, has properties of a retrotransposon.

Sigma is a 340- or 341-base pair repetitive element which is located almost exclusively within 19 base pairs of the 5' ends of various tRNA genes in the Saccharomyces cerevisiae genome. Although most sigma elements characterized to date are isolated insertions, a few of the elements occur relatively closely spaced. One of these pairs is a direct repeat of the sigma element separated by an internal domain 4.7 kilobase pairs in length. Not only does this structure resemble a composite transposable element, but regions within the sigma elements and intervening domain are homologous to conserved regions in retroviruses and retrotransposons of yeast and other organisms. Two features suggest that the sigma elements and intervening DNA transposed in a concerted event: only one of the two sigma elements is associated with a tRNA gene, and only the outside ends of the two elements are flanked by the 5-base pair direct repeats that usually flank individual sigma insertions. Examination of genomic DNA from five laboratory strains indicates that the 4.7 kilobase pair internal domain is present in one to four copies per haploid genome and that the genomic location of this domain differs from strain to strain. In addition, Northern blot analysis showed the presence of a 5.2 kilobase poly(A) transcript which hybridizes to both sigma and internal domain-specific probes. The existence of this composite element may suggest new ways to consider the mechanisms by which retrotransposons select their targets.

Base Sequence↗