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Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.
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PMID:DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA. 99 11

A cytochemical and electron microscope study has been made of leaves of sugar beet infected with beet yellows virus. Inclusions of particles, which agree in size with beet yellows virus particles isolated by other investigators, have been localized in the ground cytoplasm, in the chloroplasts, and in the nuclei. These particles are circa 100 A in diameter and have an electron-transparent core of 30 to 40 A. Use of acridine orange, azure B, and pyronine Y has revealed that the cytoplasmic inclusion bodies, which consist wholly of the elongate particles, have a strong RNA reaction removable by RNase pretreatment. Particles observed in the chloroplasts may or may not be associated with lipid spheres. If they are, the particles are confined to the periphery of the spheres. In this position the particles are arranged tangentially and are further arranged parallel into groups which lie at various angles to one another. Within the groups the particles are regularly spaced in a three dimensional lattice. Particles located free in the stromal regions are often arranged regularly in curved rows which lie parallel to one another so that a three dimensional lattice is formed. The dispersed and compact forms of virus inclusions are described and related to the condition of the associated cytoplasm. The ground cytoplasm of cells associated with the sieve elements contains numerous ribosomes. A decrease in the number of ribosomes is concomitant with the increase in size of virus aggregations in a cell. Vesiculation of some component of the cytoplasm occurs during the period of virus replication. The vesicles are approximately 100 mmicro in diameter and could be derived from the dictyosomes. At later stages of infection these vesicles collapse and convoluted membranous material appears.
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PMID:Ultrastructural features of Beta leaves infected with beet yellows virus. 597 43

Heat shock induces changes in G1 CHO cell nuclear matrix (NM) ultrastructure that may be related to heat-induced nuclear protein accumulation (Wachsberger and Coss, 1993, J. Cell. Physiol., 155:615-634). The present study quantitates recovery of alterations in NM fine structure in CHO cells heated in G1 and compares structural recovery with recovery of bulk RNA synthesis and surviving fraction (SF). Morphology of NM preparations was quantified 30 min and 20 hr following heat shock by 1) measurement of the number of fiber anastomosing points per unit area per NM, and 2) measurement of the length of fibers between points of anastomoses within individual NMs. Architectural recovery was nearly complete within 20 hr in cells heated at 43 degrees C or 45 degrees C with SFs of 0.27 or greater. No recovery of architecture was observed in heated cells with SFs of approximately 0.01 or less. The residual damage to NMs was associated with RNA-containing fiber networks as determined by means of RNase gold labeling. Recovery from inhibition of RNA synthesis following heat shock was related to recovery of NM architecture. It is suggested that 1) repair of NM architecture does not require full recovery of bulk RNA synthesis, and 2) partial or complete irreversible collapse of the NM may be responsible, in part, for heat-induced, interphase cell death.
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PMID:Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock. 751 8

Pressure denaturation of Escherichia coli ribonuclease HI (RNase HI) was studied by Fourier transform infrared (FTIR) and two-dimensional NMR spectroscopy at pD* 3.0 and 25 degrees C. A reversible transition in the pressure range of 0.1-1090 MPa was observed with second-derivative FTIR experiments. A cooperative and gradual denaturation, involving both the secondary and tertiary structures, was observed between 240 and 450 MPa. The two peaks at 1629 and 1652 cm(-1), due to beta-strands and alpha-helices, respectively, did not fully disappear after the denaturation, and are different from the spectra of the random coil peptides. The hydrogen-deuterium exchange rates of the individual backbone amide protons were determined by heteronuclear NMR combined with the pressure-jump technique at 500, 650, and 850 MPa. Although most of the amides protected in the native structure are also highly protected in the pressure-denatured state, the rate constants (0.048 +/- 0.007 min(-1)) for the amide protons at 500 MPa are similar regardless of their locations, which is an indication of the EX1 mechanism of hydrogen-deuterium exchange. The pressure-denatured state of RNase HI at 500 MPa represents a novel denatured state, which is different from a typical molten globule state at atmospheric pressure (0.1 MPa), from the viewpoint of the homogeneous rate constants. The observations at 650 MPa are essentially the same as those at 500 MPa. However, at 850 MPa, the amide exchange rates for the highly hydrophobic C-terminal half of alpha-helix I are significantly slower than those for the other part of the protein, which can be interpreted as a hydrophobic collapse centered at the C-terminal half of alpha-helix I.
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PMID:Pressure-denatured state of Escherichia coli ribonuclease HI as monitored by Fourier transform infrared and NMR spectroscopy. 992 68

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.
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PMID:Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold. 1224 46

Pear (Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia. Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Delta psi(mit)) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Delta psi(mit) collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.
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PMID:S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro. 1878 82

The germinal vesicle of the mature Chaetopterus egg is invested by an envelope which can be seen in electron micrographs to contain "pores" in its bilaminar structure. While under continuous microscopic observation, individual germinal vesicles were isolated in various test solutions by an extremely gentle method. Repeated measurements of nuclear diameter and of optical path differences with an interference microscope provided data on changes in mass after isolation. It was found that bovine serum albumin can readily penetrate the nuclear envelope of the isolated nucleus and that there are soluble elements which rapidly diffuse out. A relatively non-diffusible mass is lost at a much slower rate, the proportion of soluble to non-diffusible mass being dependent on the ionic environment. Calcium and manganese increase the proportion of the non-diffusible mass at the expense of the soluble components, while potassium decreases it. The shape and size of the isolated nucleus is at least partially dependent on the non-diffusible mass of its interior. Digestion with trypsin causes a complete structural collapse and loss of the non-diffusible elements, along with disappearance of the nucleolus. The nucleus shrinks and becomes wrinkled. A small residual mass is left which is probably associated with the nuclear envelope. Digestion with RNase or DNase causes no detectable effect on the isolated nucleus. Micromanipulation of the isolated nucleus consistently indicates that there are strands emanating from the nucleus. They may be up to several hundred microns long, are structurally strong, and are not destroyed by trypsin, RNase, or DNase. Electron micrographs of thin sections of intact cells show that the germinal vesicle is highly irregular in outline with complex evaginations extending into the cytoplasm. With the light microscope the isolated nucleus looks spherical and smooth and no emanating strands can be seen. The nature of the strands is not known.
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PMID:Permeability and Structural Characteristics of Isolated Nuclei from Chaetopterus Eggs. 1986 59

Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant-pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection.
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PMID:Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection. 2232 69

Effect of chitosan on the mesophyll cell ultrastucture and activity of hydrolases in leaves of tobacco cv. Samsun was studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased) and, at the same time, caused some activation of lytic compartment expressed in the stimulation of the formation of dictyosomes, smooth ER elements and cytoplasmic vacuoles, which are all prominent constituents of this compartment. In biochemical experiments, it was established that chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) in the leaves as compared to untreated leaves. In some cells chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm and so on) that can be classified as a result of programmed cell death development.
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PMID:[Effect of chitosan on the cell ultrastructure and activity of hydrolases in tobacco leaves]. 2346 Oct 36

Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.
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PMID:R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli. 2382 59


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