Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body

A panel of sera from 892 autoimmune patients was screened by indirect immunofluorescence on mammalian cells. Seventy-three sera were identified that recognize the nucleolus. Three of these sera appear to stain the nucleolus in yeast, suggesting that they recognize highly conserved antigens. These three sera also immunoprecipitate mammalian U3 snRNA-containing particles, which reside in the nucleolus and have been implicated in rRNA processing. Double immunofluorescence experiments with anti-nucleolus and anti-tubulin antibodies revealed a novel form of non-random nuclear organization in yeast. The spindle pole body and the nucleolus — both of which are associated with the nuclear envelope — preferentially localize at opposite ends of the nucleus. Organization of these and other components into specific regions of the nucleus may be important for optimizing their proper function.     

Dispersion population models discrete in time and continuous in space

We analyze a discrete-time model of populations that grow and disperse in separate phases. The growth phase is a nonlinear process that allows for the effects of local crowding. The dispersion phase is a linear process that distributes the population throughout its spatial habitat. Our study quantifies the issues of survival and extinction, the existence and stability of nontrivial steady states, and the comparison of various dispersion strategies. Our results show that all of these issues are tied to the global nature of various model parameters. The extreme strategies of staying-in place and going-everywhere-uniformly are compared numerically to diffusion strategies in various contexts. We approach the mathematical analysis of our model from a functional analysis and an operator theory point of view. We use recent results from the theory of positive operators in Banach lattices.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.     

Mechanism of interaction between Ku protein and DNA

The mechanism of interaction between the Ku autoantigenic protein, a heterodimer of noncovalently linked 70,000- and 80,000-dalton subunits, and DNA was studied using immunoaffinity-purified Ku protein and a 300-base pair EcoRI fragment from HeLa cell DNA. In the nitrocellulose filter-binding assay, the Ku protein bound 32P-labeled double-stranded DNA, and much less efficiently single-stranded DNA. The binding of Ku to DNA was dependent on ionic strength and prevented by IgG from patient sera containing anti-Ku antibodies. In competitive assays, using unlabeled nucleic acid competitors, the DNA binding of Ku was not inhibited in the presence of yeast tRNA, synthetic copolymer of poly(A)-poly(dT), or circular plasmid pBR322 DNA, but was inhibited when the plasmid DNA was cleaved with appropriate restriction endonucleases. The inhibitory activities of cleaved plasmid DNA were independent of the configuration or nucleotide sequences at ends but proportional to the number of recognition sites of restriction enzymes used. Footprint analysis demonstrated that Ku protein protected both 3'- and 5'-terminal regions of double-stranded DNA from DNase I digestion. When Ku protein was fractionated electrophoretically, transferred to nitrocellulose filter, and probed with 32P-labeled DNA, only the 70,000-dalton subunit exhibited DNA binding. Thus, the Ku protein appears to recognize selectively ends of double-stranded DNA molecules. Possible functions of the Ku autoantigen in eukaryotic cells are discussed.     

Age, strain and species differences in circulating parathyroid hormone

A new, sensitive parathyroid hormone (PTH) radioimmunoassay that appears specific for the intact hormone, and its validation for measuring rat PTH are described. The assay is based on antibody C2-7 from chicken immunized with bovine PTH; it has a detection limit of 6 pg of bPTH per assay tube and measures basal PTH in most rats; it is responsive to provoked changes in endogenous PTH concentration, and the intra-assay and inter-assay coefficients of variation are 6.0% and 7.2%, respectively. Multiple dilutions of rat serum and parathyroid gland extract, result in competitive inhibition curves that are parallel to that of highly purified bPTH. Under our assay conditions the C2-7 antibody cross-reacts well with intact PTH but synthetic fragments of the hormone (1-34bPTH, 1-34hPTH, 28-48hPTH, 44-68hPTH, 53-84hPTH) do not depress tracer (125l-bPTH) binding to the antibody. Studies designed to validate the assay gave predictable results such as enhanced secretion of the hormone in response to EDTA infusion, and failure to detect the hormone in serum following thyroparathyroidectomy. In addition, we made the novel observation that in F344 rats circulating immunoreactive PTH increases progressively with aging.