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Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 271 records · Page 15Linked to original sources
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

Molecular structure of r(GCG)d(TATACGC): a DNA--RNA hybrid helix joined to double helical DNA.

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA--RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

DNA↗
PubMed1982

Comparison of transfer ribonucleic acid structures using cobra venom and S1 endonucleases.

Cobra venom nuclease V1, which cleaves double-stranded RNA, has been used to study the structure of four Escherichia coli tRNAs: Phe, Glu2, Leu2, and Ile1. The cleavage patterns are compared to those found for yeast tRNAPhe, the three-dimensional structure of which is known. The cleavage patterns of all the tRNA molecules are similar, and the most sensitive cleavage is found at the central base pair of the anticodon stem. Studies of E. coli tRNALeu2, which has a large variable loop, are consistent with the formation of a base-paired stem and loop structure that is not closely bound to the remainder of the molecule. A survey of the results suggests that the V1 molecule may interact with the minor groove of the double helix with an affinity for stacked bases and that it may require two or three stacked bases for optimal binding and cleavage.

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

Molecular structure of the octamer d(G-G-C-C-G-G-C-C): modified A-DNA.

The deoxynucleotide fragment d(GpGpCpCpGpGpCpC) was synthesized and crystallized, and its three-dimensional structure was determined by x-ray diffraction techniques to a resolution of 2.25 A. The molecule forms a right-handed double helix in which the two base pairs at either end of the molecule are in the conventional A-DNA conformation, while the central four base pairs have a modified form in which alternate residues have sugar conformations that are closer to those in B-DNA than in A-DNA. The molecules have an intermolecular contact in which the planar terminal guanine . cytosine base pair lies on the flat minor groove surface of the A-DNA helix.

Crystallization↗
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

The high salt form of poly(dG-dC).poly(dG-dC) is left-handed Z-DNA: Raman spectra of crystals and solutions.

The laser-Raman spectra of crystalline d(CpGpCpGpCpG) and of aqueous poly(dG-dC).poly(dG-dC) in high salt (4M NaCl) and low salt (0.1M NaCl) solutions have been measured and compared. The spectra of the crystal and the high-salt solution show a striking congruence, which indicates clearly that the high-salt form of the aqueous polymer has the left-handed Z-DNA structure of the crystalline oligomer. These two spectra differ substantially from that of the low-salt form of the polymer, which has been found previously to have spectral characteristics of the B-form of DNA. The high salt spectrum shows a unique line due to guanine residues at 625 cm-1 which should be useful for qualitative and possibly quantitative assessment of the amount of Z-structure present in a sample of DNA.

Crystallization↗
PubMed1981

Left-handed double helical DNA: variations in the backbone conformation.

Four different crystals of d(CpGpCpGpCpG) have been solved by x-ray diffraction analysis and all form similar left-handed double helical Z-DNA molecules in the crystal lattice. Two different conformations are observed for the phosphates in the GpC sequences, as the phosphates are found either facing the helical groove or rotated away from it. The latter conformation is often found when hydrated magnesium ions are complexed to a phosphate oxygen atom. These different conformations may be used when right-handed B-DNA joins left-handed Z-DNA. Atomic coordinates and torsion angles are presented for both types of Z-DNA.

DNA↗
PubMed1981

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.

Base Sequence↗
PubMed1981

Induction of the Z conformation in poly(dG-dC).poly(dG-dC) by binding of N-2-acetylaminofluorene to guanine residues.

Poly(dG-dC).poly(dG-dC) and poly(dG).poly(dC) were modified by treatment with N-acetoxy-N-2-acetylaminofluorene, and their conformations were examined by circular dichroism and susceptibility to nuclease S1 digestion. A sample of poly(dG-dC).poly(dG-dC) modified to an extent of 28% with acetylaminofluorene (AAF) at the C(8) position of the deoxyguanosine residues showed a circular dichroism spectrum that had the characteristics of the Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. A sample of poly(dG-dC).poly(dG-dC) modified only 3% by AAF showed a spectrum characteristic of the B form of DNA. However, it was converted to the Z form at ethanol concentrations lower than required to convert unmodified poly(dG-dC).poly(dG-dC) from the B to the Z form. Poly(dG).poly(dC), which does not undergo the B-to-Z transition at high ethanol concentrations, did not show any large conformational changes with high AAF modification. Susceptibility to digestion with nuclease S1 also suggested differences in the conformations of the two modified polynucleotides. Poly(dG-dC).poly(dG-dC) modified by AAF to an extent of 28% was almost completely resistant to nuclease S1 digestion. However, both poly(dG).poly(dC) and DNA modified to similar levels by AAF were highly susceptible to nuclease S1 digestion. Two different conformations for AAF-modified deoxyguanosine are proposed, depending on whether its position is in alternating purine-pyrimidine sequences or in random-sequence DNA.

2-Acetylaminofluorene↗
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↗