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Biomedical subjects

A Nordheim

Publications and source records attributed to A Nordheim.

108 records · Page 6Linked to original sources
PubMed1983

Z-DNA and the polytene chromosome.

Indirect immunofluorescence shows that antibodies specific for the Z conformation of DNA bind to D. melanogaster polytene chromosomes in a specific and reproducible pattern. In our acetic-acid-fixed preparations, the binding is predominantly to interband regions, to puffs, and to some specific bands. As might be expected, the pattern of binding can be affected by cytological procedures. Bands and interbands show significant differences in their susceptibility to these procedures. Although further study is needed, our studies give no evidence that acetic acid treatment of intact cells is capable of causing conversion of B-form and Z-form DNAs. Instead, the experiments suggest that the Z conformation is preferentially available in interbands and a few other chromosome regions.

Animals↗
PubMed1983

Z-DNA-specific antibodies in human systemic lupus erythematosus.

Naturally occurring antibodies to left-handed Z-DNA have been shown to be present in the sera of human patients with systemic lupus erythematosus (SLE). These antibodies are of two types. One type reacts with both denatured DNA and Z-DNA. The other type is specific for Z-DNA and remained in the serum after removal of the cross-reactive antibody by extensive absorption on a denatured DNA affinity column. The antibodies appear to be specific for SLE and do not appear frequently in other rheumatic diseases. The presence of the antibody in SLE is correlated with the clinical manifestations of the disease, in parallel with antibodies to native and denatured DNA.

Antibody Specificity↗
PubMed1982

Monoclonal antibodies recognize different parts of Z-DNA.

Brominated poly(dG-dC) . poly(dG-dC), which forms a stable Z-DNA helix under physiological salt conditions, was used as an antigen to produce monoclonal antibodies from mice. The anti-Z-DNA antibodies were specific for the Z conformation as was shown by direct binding studies and a competitive solid phase assay. Competition could only be seen by brominated poly(dG-dC) . poly(dG-dC) under low and high salt conditions and unmodified poly(dG-dC) . poly(dG-dC) under high salt conditions. No binding could be seen to a number of other polynucleotides, including native and denatured DNA. Among the anti-Z-DNA monoclonal antibodies, examples differed in specificity: binding of one monoclonal antibody was blocked by methylation of cytosine; whereas the other antibody reacted equally well with the unmodified polymer or methyl cytosine-containing polymer in high ionic strength. The two antibodies are specific for different localized features of Z-DNA structure.

Animals↗
PubMed1982

Negatively supercoiled plasmids contain left-handed Z-DNA segments as detected by specific antibody binding.

Negative superhelical coiling of covalently closed DNA plasmids facilitates the formation of left-handed Z-DNA segments. This was demonstrated by binding of antibodies specific for Z-DNA. Plasmid pBR322 and two derivatives from it, pLP32 and pLP014, carrying inserts of alternating CG sequences of 32 bp and 14 bp, respectively, were used. Longer inserts required less negative superhelical density to induce the B-Z transitions. Antibody binding to supercoiled plasmids was also visualized by electron microscopy. Cross-linking of the antibody to the negatively supercoiled plasmid and restriction of the DNA with restriction endonucleases demonstrated that the antibodies combine with the CG insert in pLP32. For pBR322, however, evidence suggests that the antibody combines with a section of DNA containing 14 bases with alternating purine and pyrimidine residues with one residue out of alternation: CACGGGTGCGCATG. These cross-linking studies provide evidence for the binding specificity of the anti-Z-DNA antibodies. On the basis of experimental findings, we have calculated the changes in free energy associated with B-Z transitions in superhelical plasmids.

Antibodies↗
PubMed1982

Flipping of cloned d(pCpG)n.d(pCpG)n DNA sequences from right- to left-handed helical structure by salt, Co(III), or negative supercoiling.

Negative supercoiling of plasmid DNAs containing 24--42 base pairs of alternating d(pCpG) inserts is shown to cause the flipping of the helical hand of the inserts from right to left under physiological conditions. For a negatively supercoiled DNA with a fixed linking number, this flipping reduces its superhelicity and, therefore, is accompanied by a shift of its electrophoretic mobility in agarose gel. Quantitation of the mobility shifts indicates that essentially the whole stretch of contiguous alternating d(pCpG) flips to the left-handed helical form when the negative superhelical density (specific linking difference) is greatest than or equal to 0.03.

Base Sequence↗
PubMed1982

Isolation of Drosophila proteins that bind selectively to left-handed Z-DNA.

An affinity column for isolating Z-DNA binding proteins was made by attaching brominated poly(dG-dC) to Sephadex. Proteins from Drosophila nuclei were prepared and those that could bind to Escherichia coli B-DNA were removed from the solution. The remaining proteins were passed over the Z-DNA affinity column and then eluted with NaCl. Using both direct and competitive filter binding assays, we found that the eluted proteins bind to brominated poly(dG-dC) (Z-DNA) and poly(dG-m5dC) but not to poly(dG-dC) (B-DNA), native or denatured E. coli or calf thymus DNA, or brominated oligonucleotides. The proteins also bind to negatively supercoiled plasmids carrying Z-DNA sequences but not to relaxed or linearized plasmids in which the Z-DNA conformation is no longer present. Gel analysis reveals a mixture of several large proteins up to approximately 150,000 daltons.

Binding, Competitive↗
PubMed1982

Right-handed and left-handed DNA: studies of B- and Z-DNA by using proton nuclear Overhauser effect and P NMR.

We have differentiated between syn and anti glycosidic torsion angles in nucleic acid duplexes by measuring the transient nuclear Overhauser effect (NOE) between the sugar H-1' protons and the purine H-8 and pyrimidine H-6 base protons. The transient NOE measurements demonstrate a syn glycosidic torsion angle at guanosine and an anti glycosidic torsion angle at cytidine in poly(dG-dC) in 4 M NaCl and in poly(dG-m5dC) in 1.5 M NaCl solution. These features have been observed previously in the left-handed Z-DNA conformation of (dC-dG)3 in the crystalline state. By contrast, transient NOE studies demonstrate that both guanosine and cytidine residues adopt the anti conformation about the glycosidic bond for the right-handed poly(dG-dC) and poly(dG-m5dC) conformation in a low-salt solution. We have used P NMR to monitor the equilibrium between B- and Z-DNA forms of poly(dG-dC) in LiCl solutions; at high temperatures, the equilibrium shifts from B- to Z-DNA.

DNA↗
PubMed1981

Antibodies to left-handed Z-DNA bind to interband regions of Drosophila polytene chromosomes.

Antibodies which are specific to the Z-DNA conformation have been purified and characterized on the basis of their binding to three different DNA polymers which can form this left-handed helix. These antibodies bind specifically to polytene chromosomes of Drosophila melanogaster as visualized by fluorescent staining. The staining is found in the interband regions and its intensity varies among different interbands in a reproducible manner. This is the first identification of the Z-DNA conformation in material of biological origin.

Animals↗
PubMed1981

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.

Two cloning vector plasmids, pHSG415 (7100 bp) and a lambda phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of "biological containment". The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4--6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37 degrees C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: EcoRI, XhoI, XmaI, HindIII and PstI; pHSG415 additionally contains single BamHI, BstEII and HincII sites and may also be used to clone PvuI-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of BamHI and BstEII, is located within one of these antibiotic-resistance genes.

Anti-Bacterial Agents↗
PubMed1981

Antibodies specific for left-handed Z-DNA.

We prepared a brominated poly(dG-dC).poly(dG-dC) which forms a stable Z-DNA helix under physiological salt conditions. Rabbits and mice were immunized with brominated and unbrominated poly(dG-dC).poly(dG-dC) complexed with methylated bovine serum albumin. Antibodies specific for Z-DNA were produced. These antibodies were found not only in the sera of animals immunized with the low-salt stabilized Z-DNA [Br-poly(dG-dC).poly(dG-dC)] but also in sera from animals immunized with the unbrominated B-DNA form of the polymer. From this it is inferred that the unbrominated poly(dG-dC).poly(dG-dC) was partially converted to Z-DNA by its combination with the basic protein methylated bovine serum albumin. In addition to specific anti-Z-DNA antibody populations, two other interesting types of antibody populations were found. One of these reacted with both the Z and B forms of poly(dG-dC).poly(dG-dC). This antibody may be converting the polymer from the B-DNA to the Z-DNA form. The other type of antibody was specific for a B form of poly(dG-dC).poly(dG-dC) and did not react at all with the Z form. The antibodies raised to Z-DNA were shown to be highly specific for Z-DNA and did not react with B-DNA, RNA, DNA.RNA hybrids, or a number of other polynucleotides. This specificity for Z-DNA will make possible their use as reagents for determining the presence of Z-DNA in biological systems. Sera of autoimmune lupus mice were also shown to have a considerable amount of naturally occurring anti-Z-DNA antibody activity.

Animals↗
PubMed1981

7-Methylguanine in poly(dG-dC).poly(dG-dC) facilitates z-DNA formation.

Poly(dG-dC).poly(dG-dC) was methylated at the N-7 position of guanine through the use of dimethyl sulfate. The conversion of poly(dG-dC).poly(dG-dC) from B-DNA to the Z-DNA form was followed by measuring both the circular dichroic spectra and changes in the absorbance ratio A295/A260. Increasing methylation steadily decreases the amount of NaCl or MgCl2 that is required to convert the polymer from B-DNA to Z-DNA. At 100% methylation of the guanine residues, the modified polymer is fully converted to Z-DNA in a physiological salt solution. Kinetic experiments show that methylation markedly accelerates the speed of the conversion from B-DNA to Z-DNA in the presence of added salt. These effects may be partly due to the positive charge on guanine that accompanies N-7 methylation.

Base Sequence↗
PubMed1980

Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid.

A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.

Conjugation, Genetic↗
PubMed1979

Plasmid replication functions. III. Origin and direction of replication of a "mini" plasmid derived from R6-5.

Replicating DNA molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromide-caesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the origin-proximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated DNA fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin DNA sequence.

DNA Replication↗
PubMed1978

Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5.

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

Anti-Bacterial Agents↗
PubMedDate not supplied

Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences.

Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

Animals↗
PubMedDate not supplied

Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF.

A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter. Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62. Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE. Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE. Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins. Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80. But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1. The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF. Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control.

Animals↗