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Biomedical subjects

A Nordheim

Publications and source records attributed to A Nordheim.

At least 91 records · Page 5Linked to original sources
PubMed1987

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.

Animals↗
PubMed1986

Conformational DNA transition in the in vitro torsionally strained chicken beta-globin 5' region.

A sequence of 86 bp within the 5' region of the adult chicken beta-globin gene was found to undergo a DNA conformational transition at elevated levels of negative superhelical stress (- sigma = 0.068). In vitro chemical DNA modification studies which detect purine hyperreactivity (HR) to the alkylating agent diethyl pyrocarbonate (DEP) have identified this 86 bp long DEP-HR element. The DEP-HR element is composed of small, tandem segments with imperfect purine-pyrimidine alternations. Methylation of cytosines within GCGC sequences of the DEP-HR element facilitates this structural change. The binding of a monoclonal anti-Z-DNA antibody to the element has been revealed by chemical footprinting with DEP. These data suggest that the DEP-HR sequence can undergo a conformational transition to Z-DNA. It is unknown whether the conformational flexibility observed here occurs in vivo.

Animals↗
PubMed1986

Rate of B to Z structural transition of supercoiled DNA.

The forward rate of the B to Z transition induced by negative supercoiling of plasmid DNA containing an alternating C-G sequence has been examined using the binding of Z-DNA-specific antibodies to follow the transition. DNA samples of a plasmid containing a d(pCpG)16 X d(pCpG)16 insert were supercoiled to different extents and appropriate amounts of ethidium were bound to the DNAs to relax them and to keep the alternating C-G sequence in the right-hand helical form. Following the rapid removal of ethidium by passage through a column of cation exchange resin, the DNA becomes negatively supercoiled, which induces the flipping of the helical hand of the C-G insert. The rate of the transition is strongly dependent on the degree of supercoiling. The transition is complete in less than 50 seconds for a DNA with a specific linking difference (superhelical density) sigma of -0.09. For the same DNA, the half-time of the transition is about two minutes at sigma = -0.07 and about a factor of 10 slower at sigma = -0.05.

DNA, Superhelical↗
PubMed1986

Chemical footprinting of the interaction between left-handed Z-DNA and anti-Z-DNA antibodies by diethylpyrocarbonate carbethoxylation.

Diethylpyrocarbonate (DEPC) carbethoxylates Z-DNA to an increased extent because the reactive N-7 atoms of purine residues appear structurally more accessible on Z-DNA as opposed to B-DNA. This chemical probe was used in DEPC footprinting experiments, which confirm the specificity of binding of anti-Z-DNA monoclonal antibodies and which probe regions of close contact in this DNA-protein complex. Antibody binding to segments of Z-DNA existing in supercoiled plasmids resulted in specific protection from DEPC hyper-reactivity within the Z-DNA segment and induction of hyper-reactivity in purines lying adjacent to the Z-segment. Two different monoclonal immunoglobulin preparations, Z22 and Z44, are shown to generate specific and distinct footprint patterns when bound to the Z-helix. Binding of these antibodies was also found to affect DNA conformation within the Z-DNA segment by influencing the equilibrium between the B- and Z-helical conformations.

Antibodies, Antinuclear↗
PubMed1986

Diethyl pyrocarbonate: a chemical probe for DNA cruciforms.

Two palindromic DNA sequences were analyzed with respect to their chemical reactivities with diethyl pyrocarbonate. In negatively supercoiled plasmid templates enhanced N7 carbethoxylation was found with individual purines located in presumptive single-stranded loops of DNA cruciform structures. No enhanced reactivity at these positions was observed in linear, relaxed or low superhelical density plasmids. Hyperreactivity was found over a narrow region only, indicating that stable cruciforms contain loops of minimal size. No enhanced chemical reactivity was found with the four-way junction at the base of cruciforms. Diethyl pyrocarbonate has proved a sensitive structural probe for the analysis, with single nucleotide resolution, of DNA cruciform structures.

Base Sequence↗
PubMed1986

Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides.

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.

Animals↗
PubMed1986

DNA topoisomerase II cleaves at specific sites in the 5' flanking region of c-fos proto-oncogenes in vitro.

We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c-fos expression.

Base Sequence↗
PubMed1985

DNA intercalators induce specific release of HMG 14, HMG 17 and other DNA-binding proteins from chicken erythrocyte chromatin.

Chicken erythrocyte nuclei were incubated with DNA intercalating agents in order to isolate from chromatin specific DNA-binding proteins whose binding specificity may be determined by DNA secondary and/or tertiary structure. The intercalating agents ethidium bromide (EtBr) and propidium iodide induce the specific release of high mobility group proteins HMG 14 and HMG 17 under low ionic strength conditions. Chloroquine (CQ) intercalation also results in the selective liberation of HMG 14 and HMG 17, but, in addition, selectively releases other nuclear proteins (including histone H1A) in a pH- and ionic strength-dependent fashion. The use of this new 'elutive intercalation' technique for the isolation and purification of 'sequence-specific' and 'helix-specific' DNA-binding proteins is suggested.

Animals↗
PubMed1984

Bromination stabilizes poly(dG-dC) in the Z-DNA form under low-salt conditions.

Using circular dichroism studies, Pohl & Jovin (1972) [Pohl, F.M., & Jovin, T.M. (1972) J. Mol. Biol. 67, 375-396] demonstrated that poly(dG-dC) undergoes a salt-dependent conformational change characterized by a spectral inversion. The low-salt form corresponds to the right-handed B form of DNA and the high-salt form to the left-handed Z-DNA helix. Modification of poly(dG-dC) by adding bromine atoms to the C8 position of guanine and the C5 position of cytosine residues stabilized this polymer in the Z-DNA form under low-salt conditions. The guanine residues were found to be twice as reactive as the cytosine residues. With a modification of 38% Br8G and 18% Br5C, the polymers formed a stable Z-DNA helix under physiological conditions. The bromination produced spectroscopic features very similar to poly(dG-dC) in 4 M NaCl. However, bromination did not freeze the Z structure as was shown by ethidium bromide intercalation studies. Addition of the dye favored an intercalated B-DNA form. The conversion of B- to Z-DNA leads to profound conformational changes which were also seen by a reduced insensitivity to various exo- and endonucleases. Comparative studies showed that the brominated polymers have a high affinity to nitrocellulose filters. In 1 M NaCl, there was virtually no binding of B-DNA, but a substantial binding of Z-DNA was found even at rather low levels of bromination.(ABSTRACT TRUNCATED AT 250 WORDS)

Bromine↗
PubMed1983

Salt-induced conversion of B-DNA to Z-DNA inhibited by aflatoxin B1.

The carcinogen aflatoxin B1 was reacted with a polymer of alternating deoxyguanine and deoxycytosine residues to determine the effect that adduct formation has on the conversion of this polymer from the right-handed B-DNA form found at low salt concentrations to the left-handed Z-DNA form found at high salt concentrations. Reaction with aflatoxin strongly inhibited the salt-induced conversion of this polymer from B-DNA to Z-DNA. This inhibition could be detected even at relatively low binding levels.

Aflatoxin B1↗
PubMed1983

Formation of Z-DNA in negatively supercoiled plasmids is sensitive to small changes in salt concentration within the physiological range.

Negative supercoiling of the plasmid pBR322 with or without an insert of (dG-dC)n induces the formation of Z-DNA as measured by the binding of antibodies specific for Z-DNA. Increasing the concentration of Na+ (or K+) is shown to inhibit the B to Z-DNA conversion. This may be due to the effect of the cation on the B-Z junction. Using the data for B to Z-DNA conversion of the (dG-dC)n inserts, we have estimated the free energy change per base pair as well as the energy of the B-Z junction. In pBR322, a 14-bp segment [CACGGGTGCGCATG] is believed to form Z-DNA at bacterial negative superhelical densities under salt conditions which are similar to those found in vivo.

DNA↗
PubMed1983

Antibodies against Z DNA react with the macronucleus but not the micronucleus of the hypotrichous ciliate stylonychia mytilus.

Using indirect immunofluorescence, we studied the reaction of antibodies specific for left-handed Z DNA with the nuclei of the hypotrichous ciliate Stylonychia mytilus. In the vegetative cell, the macronucleus reacts strongly with these antibodies, but no reaction can be detected with micronuclei. However, an antibody that binds to denatured and right-handed B DNA reacts with both types of nuclei. No reaction of the anti-Z DNA antibody is seen in the macronuclear replication band. Digestion of macronuclei with DNAase I leads to a decrease in the anti-Z DNA antibody reaction. Some stages of the developing macronucleus were also investigated. No reaction is seen at the polytene chromosome stage, but following DNA elimination the nucleus is seen to react with the antibody.

Animals↗
PubMed1983

The sequence (dC-dA)n X (dG-dT)n forms left-handed Z-DNA in negatively supercoiled plasmids.

Z-DNA-specific antibodies have been used to demonstrate the formation of left-handed Z-DNA in sequences of (dC-dA)32 X (dG-dT)32 in negatively supercoiled plasmids. Z-DNA was found to form at physiological negative superhelical densities (sigma = -0.05) in the absence of high salt concentration or chemical modifications. The binding site of the antibody to the DNA sequences was demonstrated by crosslinking experiments. (dC-dA)n X (dG-dT)n sequences are widespread in eukaryotic DNA, and possible roles for such Z elements in chromatin activation or genome rearrangements are suggested.

Antibody Specificity↗
PubMed1983

Stabilization and detection of natural left-handed Z-DNA.

Left-handed Z-DNA is favored by nucleotide sequences with alternating purines and pyrimidines. The structural basis of this preference is described, as well as the factors which stabilize Z-DNA. The most important biological factors stabilizing Z-DNA are negative supercoiling and Z-DNA binding proteins. Experiments are described which make it possible to determine the nucleotide sequences of segments which form Z-DNA through the use of specific antibodies. Parts of the plasmid pBR322 form Z-DNA upon negative supercoiling. Similarly, three segments of the SV40 simian tumor virus form Z-DNA. They are located in the transcriptional enhancer region and may function, together with Z-DNA binding proteins, in facilitating the attachment of RNA polymerase to the DNA in chromatin. This is the first specific example suggesting a role of Z-DNA in regulating transcription.

Base Sequence↗